Microfluidic Chemical Reaction Circuits

ABSTRACT

New microfluidic devices, useful for carrying out chemical reactions, are provided. The devices are adapted for on-chip solvent exchange, chemical processes requiring multiple chemical reactions, and rapid concentration of reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/633,121, filed Dec. 3, 2004, and Provisional Application No.60/721,607, filed Sep. 29, 2005, the entire contents of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Work described herein has been supported, in part, by the Department ofEnergy (DOE grant no. DE-FC02-02ER63420-S-106,907) and the NationalCancer Institute (5P50 CA086306). The United States Government may havecertain rights in the invention.

FIELD OF THE INVENTION

The present inventions relate to microfabricated devices and methods forchemical synthesis using such devices. The inventions find applicationin the fields of microfluidics and synthetic chemistry.

BACKGROUND OF THE INVENTION

Microfluidic devices and methods are of significant and increasingimportance in biomedical and pharmaceutical research. However,considerable challenges remain in applying microfluidic technology tosequential syntheses of fine chemicals and pharmaceuticals. Continuousflow microreactors have recently been used to manipulate individualchemical processes on nanoliter (nL) to microliter (μL) scales withadvantages of enhanced heat transfer performance, faster diffusion timesand reaction kinetics, and improved reaction product selectivity (deMello et al., 2002, Lab on a Chip 2:7n; Kikutani and Kitamori, 2004,Macromolecular Rapid Communications 25:158; Jahnisch et al., 2004,Angewandte Chemie-International Edition 43:406; Fletcher et al., 2002,Tetrahedron 58:4735; Worz et al., 2001, Chemical Engineering Science 56,1029; Watts et al. 2003, Current Opinion in Chemical Biology 7:380).However, in multi-step procedures, flow-through systems are plagued bycross contamination of reagents from different steps; side reactions andpoor overall yield result from the inability to confine each individualstep. Improved methods and devices are needed.

A compelling application for microfluidic synthesis is in thepreparation of organic compounds bearing short-lived isotopes, whoseemission permits detailed mapping of biological processes in livingorgans. See Phelps, 2000, Proc. Nat. Acad. Sci. USA 97: 9226. Thedevelopment of sensitive radiolabeled molecular probes is crucial forexpanding the capability of target-specific in vivo imaging forbiological research and drug discovery. The United States already has avast network of PET cyclotron production sites in place as convenientsources for radiolabeled precursors (e.g., [¹⁸F]fluoride, [¹¹C]CO₂ and[¹¹C]MeI) and a few labeled biomarkers. The capacity for diversifyingradiolabeled probe structure is therefore limited only by the cost,speed, and efficiency of synthetic methods. A microfluidic device thatcould be used for synthesis of radiopharmaceuticals would constitute asignificant advance in medicine and would provide immediate andsignificant benefit to patients.

BRIEF SUMMARY

In one aspect, the invention provides a method for solvent exchangeusing a microfluidic device by i) providing a microfluidic devicecomprising a reactor, where the reactor (a) is configured to fluidicallycommunicate with at least one microfluidic channel; (b) is configured tobe fluidically isolated; and (c) is defined by a wall at least a portionof which is permeable to a gas but substantially impermeable to a liquidcorresponding to the gas; ii) introducing into the reactor a firstsolvent system comprising a first reactant; iii) fluidically isolatingthe reactor and withdrawing some or all of the first solvent system fromthe fluidically isolated reactor while retaining the first reactant inthe reactor; iv) introducing into the reactor a second solvent systemdifferent from the first solvent system.

In one aspect, the invention provides a method for removing a solventsystem from a microfluidic reactor by (i) providing a microfluidicdevice comprising a reactor, where the reactor (a) is configured tofluidically communicate with at least one microfluidic channel; (b) isconfigured to be fluidically isolated; and (c) is defined by a wall atleast a portion of which is permeable to a gas but substantiallyimpermeable to a liquid corresponding to the gas; where the reactoroptionally contains a first solvent system and, if present, the firstsolvent system comprises a first solute and optionally comprisedadditional solutes; ii) introducing into the reactor a second solventsystem comprising a second solute and optionally comprised additionalsolutes; iii) isolating the reactor, whereby the reactor contains athird solvent system and a solute denoted Solute A, where Solute A isfirst solute, the second solute, or a product of a reaction in whicheither or both of the first and second solutes are reactants and wherethe third solvent system is the same as the second solvent system or isa solvent system comprised of the combination of the first and secondsolvent systems; iv) withdrawing at least 25% of the volume of the thirdsolvent system from the fluidically isolated reactor, where the thirdsolvent system is withdrawn from the reactor more rapidly than Solute Ais withdrawn, and where the amount of Solute A in the reactor per unitvolume of the third solvent system in the reactor increases as the thirdsolvent system is withdrawn. In one embodiment Solute A is in solutionin the third solvent system the concentration of Solute A in the reactorincreases as the third solvent system is withdrawn.

In one aspect, the invention provides a method for carrying out achemical reaction using a microfluidic device by i) providing amicrofluidic device comprising a reactor, where the reactor (a) isconfigured to fluidically communicate with at least one microfluidicchannel; (b) is configured to be fluidically isolated; and (c) isdefined by a wall at least a portion of which is permeable to a gas butsubstantially impermeable to a liquid corresponding to the gas; ii)introducing into the reactor a first solvent system comprising a firstreactant; iii) fluidically isolating the reactor and withdrawing some orall of the first solvent system from the fluidically isolated reactorwhile retaining the first reactant in the reactor; iv) introducing intothe reactor a second solvent system comprising a second reactant, wherethe first reactant and the second reactant are compounds that chemicallyreact, under reaction conditions, to generate a product. The method mayinclude the further steps of fluidically isolating the reactor andmaintaining the reactor in a fluidically isolated state for a time andunder conditions sufficient for a first reaction product to accumulatein the reactor and/or may include the further step of withdrawing someor all of the reaction solvent system from the fluidically isolatedreactor while retaining the product in the reactor.

The method can include i) fluidically joining the reactor and amicrofluidic channel; ii) introducing into the reactor a third solventsystem comprising a third reactant and/or a catalyst, while retainingthe first product in the reactor; iii) maintaining the reactor in afluidically isolated state for a time and under conditions sufficientfor a second reaction product to accumulate in the reactor. Anaforementioned method can include i) fluidically joining the reactor anda microfluidic channel; ii) introducing into the reactor a third solventsystem comprising a third reactant and/or a catalyst, while retainingthe first product in the reactor; iii) maintaining the reactor in afluidically isolated state for a time and under conditions sufficientfor a second reaction product to accumulate in the reactor.

In one aspect, the invention provides a method for carrying out achemical reaction using a microfluidic device by i) providing amicrofluidic device comprising a reactor, where the reactor (a) isconfigured to fluidically communicate with at least one microfluidicchannel; (b) is configured to be fluidically isolated; and (c) isdefined by a wall at least a portion of which is permeable to a gas butsubstantially impermeable to a liquid corresponding to the gas; ii)introducing into the reactor a first solvent system comprising a firstreactant; iii) introducing into the reactor a second solvent systemcomprising a second reactant, where the first reactant and the secondreactant are compounds that chemically react, under reaction conditions,to generate a product; iv) fluidically isolating the reactor, wherebythe reactor contains (1) a reaction solvent system and (2) the first andsecond reactants and/or the product. This method may include the stepsof v) maintaining the reactor in a fluidically isolated state for a timeand under conditions sufficient for a first reaction product toaccumulate in the reactor; and withdrawing some or all of the reactionsolvent system from the fluidically isolated reactor while retaining theproduct in the reactor. An aforementioned method can include i)fluidically joining the reactor and a microfluidic channel; ii)introducing into the reactor a third solvent system comprising a thirdreactant and/or a catalyst, while retaining the first product in thereactor; iii) maintaining the reactor in a fluidically isolated statefor a time and under conditions sufficient for a second reaction productto accumulate in the reactor. In one embodiment of this method, asubstantial amount of the reaction product is produced prior to step(iv). In another embodiment of this method an insubstantial amount ofthe reaction product is produced prior to step (iv).

In one aspect, the invention provides a method for carrying out achemical reaction in an integrated microfluidic device by i) providing amicrofluidic device comprising a reactor where the reactor (a) isconfigured to fluidically communicate with at least one microfluidicchannel; (b) is configured to be fluidically isolated; and (c) isdefined by a wall at least a portion of which is permeable to a gas butsubstantially impermeable to a liquid corresponding to the gas; ii)reacting a first reactant and a second reactant in the reactor, wherethe first and second reactants are in solution in a reaction solventsystem, where the reactor is fluidically isolated, and where a firstreaction product is produced; iii) evaporating at least a portion of thereaction solvent system from the fluidically isolated reactor; iv)introducing into the reactor a solution comprising a third reactantand/or a catalyst, while retaining the first product in the reactor.

In one aspect, the invention provides a method for carrying out achemical reaction using a microfluidic device by i) providing amicrofluidic device comprising a reactor, where the reactor (a) isconfigured to fluidically communicate with at least one microfluidicchannel; (b) is configured to be fluidically isolated; and (c) isdefined by a wall at least a portion of which is permeable to a gas butsubstantially impermeable to a liquid corresponding to the gas; ii)introducing into the reactor a first solvent system comprising a firstreactant; iii) introducing into the reactor a second solvent systemcomprising a second reactant, where the first reactant and the secondreactant are compounds that chemically react, under reaction conditions,to generate a product; iv) fluidically isolating the reactor, wherebythe reactor contains 1) a reaction solvent system and 2) the first andsecond reactants and/or the product; where the reactor is coin-shapedand/or where vent channels are positioned adjacent over the reactor.

In one aspect, the invention provides a method for carrying out achemical reaction in an integrated microfluidic device by (i) providinga microfluidic device comprising a reactor and a separation columncomprising a stationary phase; (ii) introducing into the separationcolumn a solution containing a first reactant, and adsorbing the firstreactant to the stationary phase; (iii) eluting the first reactant fromthe stationary phase; (iv) introducing the first reactant into thereactor; (v) introducing the second reactant into the reactor, where thesecond reactant is introduced before, after, or simultaneously with thefirst reactant; (vi) maintaining the reactor for a time and underconditions sufficient for the first reagent and the second reagent toreact and produce a first reaction product. In some embodiments, thereactor a) is configured to fluidically communicate with at least onemicrofluidic channel; b) is configured to be fluidically isolated; andc) is defined by a wall at least a portion of which is permeable to agas but substantially impermeable to a liquid corresponding to the gas.

In one aspect, the invention provides a method for carrying outsequential chemical reactions using an integrated microfluidic device byi) providing a microfluidic device comprising a reactor and providingreagents sufficient for carrying out at least two sequential chemicalreactions; ii) carrying out a first chemical reaction in the reactor,thereby producing a product; iii) carrying out a second chemicalreaction in the reactor, where the product from (ii) is a reactant inthe second chemical reaction and where the product from (ii) is notremoved from the reactor prior to step (iii).

In certain embodiments of the aforementioned methods, the first reactantor the second reactant is purified or concentrated in an on-chipmicrofluidic separation column prior to being introduced into thereactor. In certain embodiments the separation column is an ion exchangecolumn, such as an ion exchange column that binds the a reactant. In anembodiment the separation column is an anion exchange column and thefirst reactant is ¹⁸F[fluoride]. In certain embodiments the separationcolumn is a sieve column. In certain embodiments the first or secondreactant is first bound to the stationary phase of the column in abinding step and then eluted from the stationary phase of the column inan elution step prior to being introduced into the reactor. In certainembodiments the microfluidic device has a closed flow path defined bythe separation column and one or more flow channel(s) and the bindingstep comprises circulating a solution comprising the first or secondreactant through the column at least twice. In some cases themicrofluidic device has a closed flow path defined by the separationcolumn and one or more flow channel(s) and the eluting step comprisescirculating an elution solution through the column at least twice.

In certain embodiments of the aforementioned methods, the first andsecond chemical reactions are carried out in different solvent systems.In certain embodiments of the aforementioned methods, the first solventsystem and second solvent system are introduced simultaneously. Incertain embodiments of the aforementioned methods, the reactor is not aclosed loop. In certain embodiments of the aforementioned methods, thereactor is coin-shaped.

In certain embodiments of the aforementioned methods, the reactor has afluid capacity of at least 4 ul. In certain embodiments of theaforementioned methods, the reactor is heated to produce reactionconditions that result in generation of the product. For example, insome cases the reactor contains a reaction solvent system and thereaction solvent system is heated to a temperature higher than thenormal atmospheric boiling point of the reaction solvent system.

In certain embodiments of the aforementioned methods, the reactor isconfigured to fluidically communicate with at least one flow channelthat is a distribution manifold. In certain embodiments of theinvention, such as aspects and embodiments above, the microfluidicdevice reactor is not configured be fluidically isolated and/or is notdefined by a wall at least a portion of which is permeable to a gas butsubstantially impermeable to a liquid corresponding to the gas.

In one aspect, the invention provides a method for carrying out a seriesof chemical reactions using a microfluidic device by i) providing amicrofluidic device comprising a reactor, where the reactor (a) isconfigured to fluidically communicate with at least one microfluidicchannel; (b) is configured to be fluidically isolated; and (c) isdefined by a wall at least a portion of which is substantiallyimpermeable to liquid water and liquid acetonitrile, but permeable towater vapor and acetonitrile vapor; ii) introducing into the reactor anaqueous solution comprising [¹⁸F]fluoride; iii) introducing into thereactor an acetonitrile solution comprising mannose triflate; iv)fluidically isolating the reactor; v) reacting the [¹⁸F]fluoride and themannose triflate to produce2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose; vi)fluidically joining the reactor and a microfluidic channel; vii)introducing aqueous HCl into the reactor while retaining the2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose in thereactor; viii) fluidically isolating the reactor; ix) hydrolyzing the2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose to produce18F-FDG. In some embodiments, the method includes (a) introducing[¹⁸F]Fluorine into the reactor in an aqueous solvent system; (b)removing the aqueous solvent system and replacing it with acetonitrile;and (c) introducing an acetonitrile solution of mannose triflate intothe reactor, prior to step (ii). In some embodiments the microfluidicdevice includes a microfluidic separation column; has a closed flow pathdefined by the separation column and one or more flow channel(s); andthe binding step includes circulating a solution comprising the first orsecond reactant through the column at least twice.

The method of claim where the [¹⁸F]fluoride is first bound to thestationary phase of the column in a binding step and then eluted fromthe stationary phase of the column in an elution step prior to beingintroduced into the reactor, and where the binding step comprisescirculating a solution comprising the first or second reactant throughthe column at least twice and/or the eluting step comprises circulatingan elution solution through the column at least twice.

In another aspect, the invention provides a microfluidic device thatincludes a separation column comprising an immobile phase through whicha fluid can pass, the column having an inlet and an outlet; and one ormore flow channel(s) not comprising the solid phase; where the flowchannel(s) and separation column define a closed path. In someembodiments the device includes a peristaltic pump capable of movingfluid through the closed path. In some embodiments the device includes areactor configured to be in fluidic communication with one of the one ormore flow channels.

In another aspect, the invention provides a microfluidic device having areactor, where the reactor i) does not form a closed path; ii) can befluidically isolated; iii) has a liquid capacity of from 5 microlitersto 10 microliters. In some embodiments the device has from 1 to 5reactors. In some embodiments the device has a single reactor. In someembodiments the device has a coin-shaped reactor. In some embodimentsthe device has a reactor is configured to fluidically communicate withat least one flow channel that is a distribution manifold.

In one aspect, the invention provides a method for removing solvent froma reaction chamber (reactor) of a microfluidic device, the method byproviding a microfluidic device comprising a reactor that contains asolute compound and a solvent system and removing all or a portion ofthe solvent system while retaining the solute compound in the reactor,whereby the amount of the solute compound per unit volume of the solventis increased. In some embodiments the solute compound remains insolution and the concentration of the solute in the solution isincreased. In some embodiments at least 50% of the solvent system isremoved from the reactor. In some embodiments at least 95% of thesolvent system remains in the reactor. In some embodiments the solventis water while in others the solvent is other than water. In someembodiments the solute compound comprises a radionuclide or a moleculecomprising a radionuclide. For example, the radionuclide is [¹¹C],[¹²⁴I], [¹⁸F], [¹²⁴I], [¹³N], [⁵²Fe], [⁵⁵Co], [⁷⁵Br], [⁷⁶Br], [⁹⁴Tc],[¹¹¹In], [⁹⁹Tc], [¹¹¹In], [⁶⁷Ga], [¹²³I], [¹²⁵I], [¹⁴C], or [³²P]. In anembodiment the solute compound is [¹⁸F]fluoride or [¹⁸F]-potassiumfluoride. In an embodiment the solute compound is i)2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose; ii)2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrile;or iii) D-mannose triflate. In one embodiment the solute compound is acryptand, for example 4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane.

In one aspect, the invention provides a method for synthesizing aradiolabeled product in a microfluidic environment by mixing aradiolabled reactant with a precursor reactant compound to produce aradiolabeled product, where the mixing and reacting occurs in amicrofluidic reactor and where the radiolabled reagent is introducedinto the reactor in a first solvent and the radiolabeled precursor isintroduced in a second solvent that is different from the first. In anembodiment the radiolabeled reactant is [¹⁸F]-potassium fluoride and theprecursor reactant is2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrileor D-mannose triflate. In an embodiment, the radiolabeled product is aradiolabeled molecular imaging probe. In an embodiment the precursorreactant is D-mannose triflate;2-(1-{6-[(2-[(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-naphthyl}ethylidine)malononitrile;N-Boc-5′-O-dimethoxytrityl-3′-O-(4-nitrophenylsulfonyl)-thymidine;N2-(p-anisyldiphenylmethyl)-9-[(4-p-toluenes-ulfonyloxy)-3-(p-anisyldiphenylmethoxymethyl)butyl]guanine;N2-(p-anisyldiphenylmethyl)-9-[[1-[(.beta.-anisy-ldiphenylmethoxy)-3-(p-toluenesulfonyloxy)-2-propoxy]methyl]guanine;8-[4-(4-fluorophenyl)-4,4-(ethylenedioxy)bu-tyl]-3-[2′-(2,4,6-trimethylphenylsulfonyloxyethyl)]-1-phenyl-1,3,8-triazas-piro[4.5]decan-4-one;5′-O-Boc-2,3′anhydrothymidine;N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-1]-4-nitro-N-2-pyridinyl-benzamide;1,2-bis(tosyloxy)ethane and N,N-dimethylethanolamine; ditosylmethane orN,N-dimethylethanol amine.

In certain embodiments, an aforementioned method includes the furtherstep of concentration of the radioactive reactant and/or deprotection orchemical modification of the radiolabeled product to produce aradiodiagnostic agent or radiotherapeutic agent.

In various aspects of the invention, a radiolabeled molecular imagingprobe or a precursor of a radiolabeled molecular imaging probe isproduced, such as 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG);6-[¹⁸F]fluoro-L-3,4-dihydroxyphenylalanine ([¹⁸F]FDOPA);6-[¹⁸F]fluoro-L-meta-tyrosine ([¹⁸F]FMT),9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine ([¹⁸F]FHBG),9-[(3-[¹⁸F]fluoro-1-hydroxy-2-propoxy)methyl]guanine([¹⁸F]FHPG),3-(2′-[¹⁸F]fluoroethyl)spiperone([¹⁸F]FESP),3′-deoxy-3′-[¹⁸F]fluorothymidine([¹⁸F]FLT),4-[¹⁸F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyr-idinyl-benzamide([¹⁸F]p-MPPF),2-(1-{6-[(2-[¹⁸F]fluoroethyl)(met-hyl)amino]-2-naphthyl}ethylidine)malononitrile([¹⁸F]FDDNP),2-[¹⁸F]fluoro-alpha-methyltyrosine, [¹⁸F]fluoromisonidazole([¹⁸F]FMISO),5-[¹⁸F]fluoro-2′-deoxyuridine([¹⁸F]FdUrd), [¹¹C]raclopride,[¹¹C]N-methylspiperone, [¹¹C]cocaine, [¹¹C]nomifensine, [¹¹C]deprenyl,[¹¹C]clozapine, [¹¹C]methionine, [¹¹C]choline, [¹¹C]thymidine,[¹¹C]flumazenil, [¹¹C]alpha-aminoisobutyric acid or a protected form ofany of the foregoing compounds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic diagram of a device for synthesis of theradioimaging agent [¹⁸F]FDG.

FIG. 2. (A) Schematic representation of a chemical reaction circuit(CRC) used in the production of 2-deoxy-2-fluoro-D-glucose (FDG) (3a,b).Five sequential processes—(i) concentration of dilute fluoride ion usinga miniaturized anion-exchange column located in a square-shaped fluorideconcentration loop, (ii) solvent exchange from water to dry MeCN, (iii)fluorination of the D-mannose triflate precursor 1, (iv) solventexchange back to water; and (v) acidic hydrolysis of the fluorinatedintermediate 2a (or 2b) in a ring-shaped reaction loop—produce nanogram(ng) levels of FDG (3a,b). The operation of the CRC is controlled bypressure-driven valves, with their delegate responsibilities illustratedby their colors: red for regular valves (for isolation), yellow for pumpvalves (for fluidic metering circulation), and blue for sieve valves(for trapping anion exchange beads in the column module). (B) Opticalmicrograph of the central area of the CRC. The various channels havebeen loaded with food dyes to help visualize the different components ofthe microfluidic chip: (red) control channels for regular valves, (blue)control channels for sieve valves, (yellow) control channels for pumpvalves, and (green) fluidic channels. Inset: Actual view of the device;a penny (18.9 mm in diameter) calibrates the dimensions on the device.

FIG. 3. Schematic representations illustrate the operation mechanisms of(A) a regular valve having a round-profiled fluidic channel and (B) asieve valve having a rectangular-profiled fluidic channel. When pressureis introduced into the control channels, the elastic membranes expandinto the fluidic channels. In a regular valve, the fluidic channel iscompletely sealed because of the perfect fit between the expandedmembranes and the round profile of the fluidic channel. In a sievevalve, the square-profiled fluidic channel is only partially closed,which allows fluid to flow through the two edges. Sieve valves can beused to confine solid objects within the fluidic channel, but allowliquid to flow through it. (C) Schematic illustration of the loading ofanion exchange beads into a column module incorporating one fluidicchannel and five sieve and five regular valves. [□], open valve; [X],closed valve. A suspended solution of anion exchange beads is introducedinto the column modules where five sieve valves and five regular valvesoperate cooperatively to trap anion exchange beads inside the fluidicchannel (total volume: 10 nL). A miniaturized anion exchange column forfluoride concentration is achieved when the fluidic channel is fullyloaded. (D) A snapshot of the bead-loading process in action.

FIG. 4. Schematic diagrams show the four most critical steps of FDG(3a,b) production in the CRC. (A) Concentration of dilute fluoride ion:with the cooperation of regular valves, a dilute fluoride solution(indicated in blue) is introduced into the ion exchange column by ametering pump. (B) Evaporating water from the concentrated KF solution:after transferring the concentrated KF solution from the fluorideconcentration loop to the circular-shaped reaction loop, the CRC isheated on a hotplate to evaporate water from the reaction loop.Meanwhile, all of the surrounding regular valves are completely closedand the circulating pump is turned on. (C) Fluorination reaction: afterintroducing a MeCN solution (green) of Kryptofix and the D-mannosetriflate 1 into the reaction loop, the inhomogeneous reaction mixturewas isolated in the reaction loop, mixed using the circulating pump, andheated under a computer-controlled gradient to generate the intermediate2a (or 2b). (D) Hydrolysis reaction: after evaporating the MeCN, an HClsolution (blue) is introduced into the reaction loop to hydrolyze theintermediate 2a (or 2b) to give the final product, FDG (3a,b).

FIG. 5. (A) Analytical TLC profile of the unpurified mixture (bluecurve) obtained upon the production of [¹⁸F]FDG (3a) in the secondgeneration CRC indicating that the radiochemical purity of the FDGproduction is up to 96.2%. The two peaks have R_(f) values of 0.0 and0.36 corresponding to [¹⁸F]fluoride and [¹⁸F]FDG (3a), respectively.After purification and sterilization, the [¹⁸F]FDG (3a) (black curve)with 99.3% radiochemical purity was employed for mouse microPET/microCTimaging. (B) Projection view of microPET/microCT image of atumor-bearing mouse injected with [¹⁸F]FDG produced in a microfluidicchip. Organs visible are the bladder, kidneys, heart, tumor and twolymph nodes.

FIG. 6. Schematic diagrams summarize the fluoride concentration processwhich consisted of 9 steps in the CRC. (A) Diluted fluoride solution(indicated in blue) was introduced into the fluoride concentration loopfrom the top-left channel on the chip. The fluoride ion was trapped byanion exchange beads in the column. The filtrate solution was exportedout of the device through the waste channel. The loading speed offluoride solution was controlled by the metering pump (labeled inyellow). The loading process took around 2 min. (B) Following fluorideloading, 18 nL of K₂CO₃ solution (0.25 M) was pumped into fluorideconcentration loop from the left-middle channel. This step takes 6seconds at 25° C. (C) The K₂CO₃ solution was circulated in the fluorideconcentration loop for 2 minutes to assure all the fluoride trapped onbeads was released into the solution. By the end of this step, thefluoride concentration within the loop can increase by two orders ofmagnitude compared to the concentration of loaded fluoride solution. (D)After circulation, 20 nL of K₂CO₃ solution was introduced into thefluoride concentration loop to displace the concentrated fluoridesolution into the reaction loop. This dead-end filling process (all thevalves are closed except the valve controlling the loading channel, theair inside the loop is pushed out through the porous PDMS matrix) took20 seconds. (E) With all the valves around reaction loop closed, the CRCwas heated on a digitally controlled hotplate with a gradient (100° C.for 30 seconds, 120° C. for 30 seconds, 135° C. for 3 minutes). Most ofthe water from the concentrated fluoride solution was removed throughdirect evaporation. (F) The CRC was cooled down to 35° C. within 1minute. (G) Anhydrous MeCN (in green) was introduced into the reactionloop through the bottom middle channel by dead-end filling. This steptook less than 20 seconds at 25° C. (H) The CRC was heated again with agradient (80° C./30 seconds, 100° C./1 minutes) to remove the remainingwater inside the loop. With all valves around the loop closed, MeCN andwater vapors were removed through direct evaporation. (I) The CRC wascooled down to 35° C. within 40 seconds.

FIG. 7. Schematic diagrams summarize the fluorine substitution processwhich is composed of 3-step sequential operations in the CRC. (A)Kryptofix 222/the mannose triflate 1 in anhydrous MeCN were introducedfrom the top middle channel to the reaction loop by dead-end filling.This step took 20 seconds at 25° C. (B) The CRC was heated with agradient (100° C./30 seconds, 120° C./50 seconds). At the same time, thesolution was actively mixed by the circulating pump. The fluorinatedintermediate 2a (or 2b) was obtained by the end of this step. (C) TheCRC was cooled down to 35° C. within 40 seconds.

FIG. 8. Schematic diagrams summarize the hydrolytic process which iscomposed of 3-steps sequential operations in the CRC. (A) HCl aqueoussolution (3.0 N) was introduced from the top right channel (in lightblue) to the reaction loop by dead-end filling. This step took 20seconds at 25° C. (B) The HCl and the fluorinated intermediate 2a (or2b) were mixed by the circulating pump for 1 minute at 60° C. In thisstep, the intermediate 2 (or 2b) was hydrolyzed to yield the finalproduct FDG (3a,b). (C) The solution containing FDG (3a,b) (in darkblue) was flushed out of the device though the product line located atthe bottom of the CRC.

FIG. 9. (A) GC-MS plot of a mixture containing MeCN, mannose triflate 1and Kryptofix 222. The two peaks at retention times of 14.73 and 18.10min correspond to mannose triflate 1 and Kryptofix, respectively. (B)GC-MS plot of the mixture in (A) after its reaction with concentratedfluoride in the CRC. The peak having a retention time of 14.16 mincorresponds the formation of the fluorinated intermediate 2b. Acalibrated integration of the chromatogram suggests a conversion yieldof 95%. (C) GC-MS plot of a TMS-functionalized [¹⁹F]FDG (3b) which isobtained by treating crude [¹⁹F]FDG (3b) with TMSCl. The calibratedintegration indicates that the hydrolytic reaction of intermediate 2bresulted [¹⁹F]FDG (3b) in >90% purity.

FIG. 10. Analytical TLC profile of the unpurified mixture obtained uponthe sequential production of [¹⁸F]FDG (3a) in the first generation CRCindicating that the radiochemical purity of the FDG production is up to97.3%. The two peaks with values for R_(f) of 0.0 and 0.4 correspond to[¹⁸F]fluoride and [¹⁸F]FDG (3a), respectively.

FIG. 11. (A) A photograph of second generation CRC in use for [¹⁸F]FDGproduction. (B) Schematic representation of the second generation CRCcomposed of three major functional components, including (i) ventchannel, (ii) coin-shaped reactor and (iii) manifold for introduction ofthe mannose triflate solution.

FIG. 12 shows schematic diagrams summarizing the [¹⁸F]FDG (3a) synthesison the second generation CRC. (A) Concentrated mixture of¹⁸F⁻/Kryptofix222/K₂CO₃ in MeCN is introduced into the reaction chamberuntil the reaction chamber is ⅔ full. This process is accelerated byapplying vacuum to the vent above the reaction chamber to remove the gasbeing displaced by the fluoride solution. (B) The mannose triflate 2asolution in MeCN (25 mg/mL) is loaded to fill the distribution manifoldby dead-end filling. The manifold is designed to introduce mannosetriflate 2a solution into the reaction chamber equally andsimultaneously through the six ports. (C) The mannose triflate 2asolution is introduced into the reaction chamber by employing 10 psi ofloading pressure. (D) The reaction mixture was kept at 25° C. for 5 min,and the fluorination reaction is carried out at 65° C. for 2 min. Thevacuum in the vent is then turned on to evaporate ¼ of MeCN in thereaction chamber. (E) 3N HCl solution is loaded into the reactionchamber, and the acidic hydrolysis is performed at 60° C. (F) Theremaining MeCN is evaporated at 75° C. for 5 min. (G) The reactionchamber is cooled to 40° C. and vacuum in the vent is turned off priorto elution. (H) As the valves are opened on the water inlet and theproduct outlet, [¹⁸F]FDG (3a) is flushed out of the chamber. Thetangential inlet and outlet allow the water trajectory to follow alongthe far wall of the reaction chamber ensuring complete product elution.

FIG. 13 shows an optical micrograph of a PDMS-based integrated CRC,which is intentionally filled with different dyes in order to betterdistinguish different modules in the device. The red and yellow linessignify the control channels associated with valve and pump modules,respectively. Both of the control lines are connected with gas manifoldsand driven by gas pressures ranging between 5 and 30 psi. The blue andgreen lines indicate fluidic channels in which solutions containingstarting materials and reagents are transferred and stored. At theintersections of control (red and yellow) and fluidic (blue and green)channels, valve and pump modules are located. The deflection ofindividual valve membrane is utilized to impede fluid flow. Threeparallel-oriented valves (yellow) can be grouped to form a peristalticpump module that is employed to control both the direction and rate ofthe flow in the corresponding fluidic channels by varying the pumpsequence and period.

FIG. 14 shows the design of a device used to synthesize2-(1-(6-[(2-[18F]fluoro-ethyl)(methyl)amino]-2-naphthyl)(FDDNP). Flowchannels, including the reactor loop are shown in blue, Control channelsare shown as thin red lines. Vent channels (400 microns wide and 25microns high) are shown using thick red lines. Reagents can beintroduced in a variety of ways. Shown is one way to introduce reagentsfor FDDNP synthesis, i.e., fluoride/K2CO3 solution introduced via thetop channel, and precursor/Kryptofix solution are introduced through alower channel. MeCN can be introduced via the second line from the topwhile keeping the valve immediately above the center line closed toflush product out of the reactor and through the exit channel as shown.FIG. 14 also indicates that a lower channel can be used for introductionof HCl. The synthesis of FDDNP did not require a hydrolysis step.However, the same chip design has been used for a synthesis of3′-deoxy-3′-[18F]fluorothymidine(“[¹⁸F]FLT”) which does include an acidhydrolysis step. In that case, acid can be introduced as indicated.

FIG. 15 shows PDMS-based chemical reaction circuits for FDG synthesis.

FIG. 16. (A, top) An optical micrograph of a square shaped concentrationloop containing an anion exchange column, a pump and a concentrationloop for concentration of dilute fluoride (B, bottom) An opticalmicrograph of a round-shaped reaction chamber containing a reaction loopand a pump, used for both fluorination and hydrolysis reactions.

FIG. 17 is a graphical representation summarizing sequential FDGsynthesis in a chemical reaction circuit.

FIG. 18 shows a chip with a coin-shaped reactor and an “off-chip”chromatography column.

FIG. 19 shows a microscale reaction chamber with a large bottom-upmixer.

FIG. 20 shows a radiator mixer integrated with the radiator evaporator.

DETAILED DESCRIPTION Section 1. Definitions

As used herein, “fluid” refers to a liquid capable of flowing through amicrochannel (or “flow channel;” see description in Section 3B, below)having at least one cross-sectional dimension less than 1 mm. Forpurposes of this disclosure, the term “fluid” does not encompassesgasses.

As used herein, the terms “microfluidic device,” “integratedmicrofluidic device,” and “chip,” are used interchangeably to refer to asingle integral unit that has a microfluidic reactor, microfluidic flowchannels, and valves. Microfluidic devices typically also have othermicrofluidic components, such as pumps, columns, mixers, and the like.Most often the chip is fabricated from elastomer, glass, or silicon.Typically, the chip is box-shaped with a height that is relatively smallcompared to length and width; however, the chip can have other shapesincluding cubical, cylindrical, and others.

As used herein, the term “Chemical Reaction Circuit (CRC),” refers to achip that contains a microfluidic reactor, flow channels, and valves,and has an architecture that renders the chip useful for carrying outsequential chemical reactions.

As used herein, a “microfluidic system” refers to a system for carryingout sequential chemical reactions, and comprises at least onemicrofluidic device (e.g., CRC) as well as one or more componentsexternal to the device(s). Examples of external components includeexternal sensors, external chromatography columns, actuators (e.g.,pumps or syringes), control systems for actuating valves, data storagesystems, reagent storage units (reservoirs), detection and analysisdevices (e.g., a mass spectrophotometer), and other components known inthe art.

The terms “fluidic communication,” “configured to fluidicallycommunicate,” “configured to be fluidically isolated” “fluidicallyisolated,” and “fluidically joined,” describe relationships betweencomponents of a microfluidic system, and particularly the relationshipof flow channels and valves with a reactor, or the relationship of flowchannels and valves with a microfluidic column.

As used herein, “fluidic communication” has its usual meaning in themicrofluidic arts. Two chip components are in “fluidic communication”when a fluid can be transported (e.g., pumped) from one component to theother. For example a reactor and a flow channel are in fluidiccommunication when the flow channel connects to the reactor and anyvalve(s) that would prevent transport of a liquid from the reactor tothe channel are in an “open” position. Likewise, a reactor and columnthat are connected by a flow channel are in fluidic communication whenany valve(s) that would prevent transport of a liquid from the reactorto the column are in an “open” position.

The terms “configured to fluidically communicate” and “configured to befluidically isolated,” as used herein, refer to the presence of valvespositioned or situated to prevent or permit transport of fluid from onechip component to another. Two components are “configured to fluidicallycommunicate” if fluid could be transported from one component to theother provided any valves that would prevent flow between the componentswhen closed are open. “Configured to fluidically communicate” refers torelationship between microfluidic components that give them thepotential of being in fluidic communication, although two components“configured to fluidically communicate” may or may not be in actualfluidic communication.

A reactor is “configured to be fluidically isolated” when valves arepositioned such that, if they were closed, the reactor would not be influidic communication with any other chip component (i.e., fluid wouldbe confined to the reactor). Thus, a reactor that is “configured to befluidically isolated” has the potential (if appropriate valves areclosed) to be in fluidic communication with other chip components, suchas flow channels and has valves positioned so that, if closed, thereactor is not in fluidic communication with other components. Chipcomponents other than reactors can also be “configured to be fluidicallyisolated,” i.e., when valves are positioned such that, if they wereclosed, the component would not be in fluidic communication with anyother chip component (i.e., fluid would be confined to the fluidicallyisolated component). A reactor or other component that is configured tobe fluidically isolated is “fluidically isolated” when the reactor orcomponent is not in fluidic communication with any another component(e.g., valves are closed) and is “fluidically joined” when at least onevalve is open and the reactor or component is in fluidic communicationwith at least one another component.

The term “fluidically isolating” refers to the process in which valvesare closed (actuated) to change the state of a reactor or othercomponent from fluidically joined to fluidically isolated. The term“fluidically joining” refers to the process in which valves are openedto change the state of a reactor or other component from fluidicallyisolated to fluidically joined.

In the special case in which one-way valves are used, the terms “fluidiccommunication,” “configured to fluidically communicate,” “configured tobe fluidically isolated” “fluidically isolated,” and “fluidicallyjoined,” are intended to take into account the directionality of flow.One-way valves (e.g., one-way valves, check valves, and fluidicrectifiers or diodes) allow fluidic transport in only one direction suchas from a flow channel into a reactor but not in the other direction(see, e.g., Adams et al., 2005, J. Micromech. Microeng. 15:1517-21; andreferences 6-12 therein). For example, a reactor connected to four flowchannels, each of which is divided from the reactor by a one-way valveoriented to allow flow into but not out of the reactor, would beconsidered fluidically isolated. A reactor connected to four flowchannels, three of which were oriented to allow flow in but not out ofthe reactor, one of which was a conventional two-way valve, would beconsidered fluidically isolated when the two-way valve was closed andwould be considered fluidically joined when the two-way valve was open.In the case in which a first chip component (e.g., column) is connectedvia a flow channel to a second component (e.g., reactor) with anintervening one-way valve allowing flow only from the first to thesecond component, the first component is in fluidic communication withthe second component, but the second component is not in fluidiccommunication with the first component.

As used herein, the “reactants” are molecules that are capable ofchemically interacting with each other under suitable reactionconditions to produce a product.

As used herein, a “chemical reaction” is a process involving one, two ormore substances (reactants) in solution, that chemically interact toyield one or more product(s) which are different from the reactants.Examples of chemical interactions include molecules or radicalscombining to form larger molecules, molecules breaking apart to form twoor more smaller molecules, and rearrangements of atoms within molecules.Most often, a chemical reaction involves the breaking and creation ofcovalent bonds. As used herein, a mere change of state (e.g.,crystallization; isomerization, interconversion of polymorphs, ortransition from liquid to gas) by itself is not a chemical reaction. Inimportant embodiments, the reactant(s) and product(s) are in solutionduring the chemical reaction process. In certain embodiments, processesin which reactants are immobilized on a solid phase (e.g., conjugated toa bead) are specifically excluded from the definition of chemicalreactions. In certain embodiments, reactions catalyzed by enzymes (e.g.,proteins, ribozymes or the like) are specifically excluded from thedefinition of chemical reactions.

As used herein, two chemical reactions are “sequential” when a productof one reaction (i.e., the first reaction) is a reactant or catalyst inthe other reaction (i.e., the second reaction).

As used herein, a “chemical process” means a chemical reaction, theprocess of solvent exchange, or the process of concentration.

As used herein, the term “solvent system” refers to a solvent (e.g.,acetonitrile) or combination of solvents (e.g., 25% methanol/75% water)in which a solute is or can be dissolved.

As used herein, the term “reaction solvent system” refers to the solventsystem present in a reactor at the time, following introduction of allreactants for a particular chemical reaction, that the reactor isfluidically isolated. Thus, the reaction solvent system is comprised ofthe solvent systems in which the reactants are introduced into a reactorplus any solvent(s) present in the reactor prior to introduction of thereactants, as modified by any solvent(s) withdrawn from the reactorafter the first introduction of a reactant for the particular chemicalreaction and prior to the time the reactor is fluidically isolated.Generally, the reaction solvent system is the solvent system in which achemical reaction takes place in a reactor.

As used herein, reference to removal of a solvent system from a reactor“while retaining” a solute, reactant, product or other compound meansthat solvent is removed by evaporation from a fluidically isolatedreactor. When a solvent system is removed from a reactor while retaininga solute compound in the reactor according to the invention, the rate ofloss of solvent from the reactor exceeds the rate of loss of the solutecompound. Thus the process of removing a solvent system from afluidically isolated reactor while retaining the solute compound in thereactor results in an increase in the amount of the compound in thereactor per unit volume of the solvent in the reactor. In the absence ofcomplete removal of solvent and/or precipitation of the compound, theconcentration of the compound in the solution increases with the removalof the solvent system. In certain cases, the solvent is evaporatedwithout removing the compound from the reactor (see below). In othercases some portion of the compound in the reactor enters or passesthrough the gas permeable portion of the reactor wall (e.g., theelastomer).

As used herein, reference to removal of a solvent system from a reactor“without removing” a solute, reactant, product or other compound meansthat at most an insignificant proportion of the solute, reactant,product or other compound is removed from the reactor. In this contextan insignificant amount is less than 25%, more often less than 10%, veryoften less than 5% and sometimes less than 1% of the amount in thereactor prior to solvent removal. In some cases, no detectable amount ofthe compound is removed.

As used herein, a “closed path,” or a “closed flow path” refers to aflow channel or a combination of flow channels (including channels inwhich a chromatography material is disposed) thorough which liquid cancirculate. A closed path means that the channel or combination ofchannels can be temporarily isolated from other parts of the chip, forexample by closing valves in any channels which lead into or out of thepath, and that a liquid can then circulate through the path (whendriven, for example, by a pump). A closed flow path may be circular(see, e.g., FIG. 1), rectilinear (see, e.g., FIGS. 6C and 14),curvilinear, and the like. Examples of closed paths include loopchannels and concentration loops.

Section 2: Overview

The invention provides, in one aspect, an integrated microfluidicdevice, or chip, in which chemical reactions, and in particular,sequential chemical reactions, can be carried out. The chip has (1) atleast one reactor that is fabricated at least in part from agas-permeable material and that is configured to fluidically communicatewith microfluidic flow channels, and (2) valves sufficient tofluidically isolate the reactor. Other microfluidic components also canbe integrated into the chip, including, for example, control channels,guard channels, vent channels, fluid reservoirs, mixing reactors, rotarymixers, separation modules (e.g., separation columns), sorting regions,pumps, ports, vias, nozzles, monitoring systems, lenses, sensors,temperature control systems, heat sources, light sources, waveguides andthe like. Examples of microfluidic chips include elastomeric chips,non-elastomeric chips, and partially elastomeric chips.

The invention also provides methods for carrying out chemical processesusing a microfluidic chip and system. Using these methods, a widevariety of products can be synthesized rapidly, in high yields, and atlow cost.

Although the invention is described in detail with reference to specificembodiments below, a brief overview of an exemplary chip and method willaid the reader in understanding the invention. It will be appreciatedthat this brief description is for illustration and is not intended tolimit the invention in any way.

The chip architecture illustrated in FIG. 1 represents a design that iscapable of supporting several sequential chemical processes, includingion exchange, product purification, solvent evaporation, aqueouschemical reactions, anhydrous chemical reactions, and chemical reactionsunder elevated temperature or pressure conditions. Channel 111 can serveas a chromatographic column for the purpose of supporting the chemicalprocesses of ion exchange, or reactant or product purification, or thelike. To do this, channel 111 is loaded with an appropriatechromatographic resin material designed for ion exchange or product orreactant purification, as illustrated. Valve 110E is configured toretain a chromatographic resin but allow fluid to flow through thevalve. A reaction mixture may be flowed through the column by openingvalve 110A and introducing the reaction mixture through channel 113,with valves 110B and 110D closed.

It may be desirable to route a reaction mixture through a columnmultiple times. In that case, the reaction mixture is introduced throughchannel 113 via open valve 110A. Valve 110E and 110C are closed. The airpermeability of the channel material is then utilized to completely fillchannel 111 (column 111) and channel 112 with a reaction mixture (e.g.,comprising a reactant in solution). Peristaltic pump 103B is used tocycle the reaction mixture around the loop described by the closed andopen valves, thereby pushing the reactant mixture through the column 111multiple times.

(The same column may be alternatively used by flowing a reaction mixturethrough the column, and then using the flow-through fraction (or,alternatively an eluate) in other regions of the chemical reactioncircuit for subsequent chemical processing. For example, a reactionmixture can be introduced via column 113 through open valve 110A, withvalves 110B and 110D closed, and eluting the product through valve 110Efor further use.)

The reaction mixture prepared or purified on the column 111 may then besubjected to further chemical processes. By opening valves 110A, 110D,110C, 105A, and 102A the reaction mixture may be flushed off of thecolumn, through channel 100, and introduced into the reaction chamber104 (reactor 104). (Alternatively, a reaction mixture may be introducedinto reactor 104 from another source via channel 100.) Additionalchemical reactants may be introduced from channel 109 by opening valve105B (optionally with valve 110C closed). Other reactants may beintroduced from, for example, channel 107 by opening valve 102B. Thereaction mixture within the reaction chamber 104 may be mixed usingperistaltic pump 103C for a desired amount of time. The entire reactionmixture may be heated during the chemical reaction process by heatingthe chip or a section thereof.

Solvent may be removed from reaction chamber 104 by evaporating thesolvent through the matrix material from which the microfluidic chemicalreaction circuit, reactor, or portion thereof is comprised. Additionalsolvents and/or reagents may then be introduced into the reactorthrough, for example, channel 107.

It will be apparent that reaction chambers of various shapes and sizes,additional columns, etc., may be introduced into the chemical reactioncircuit design as necessary to accomplish specific chemical processes.

A specific embodiment of a chemical reaction circuit, designed for thepreparation of the radiolabeled molecular imaging agent,[¹⁸F]fluordeoxyglucose([¹⁸F]FDG) is presented in FIG. 2. This particularapplication of chemical reaction circuits illustrates many importantaspects of the Chemical Reaction Circuit (CRC), since the preparation of[¹⁸F]FDG involves the use of a column, multiple solvents, sequentialchemical steps, chemical processes at elevated temperatures andpressures, and product elution. In particular, the synthesis of [¹⁸F]FDGproceeds according to a synthetic scheme that includes the followingsequential chemical reactions:

¹⁸F-fluoride+mannosetriflate→2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose(¹⁸F-FTAG)  I.(fluorination)

¹⁸F-FTAG+HCl→2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose(¹⁸FDG)  II.(hydrolysis)

The device shown in FIG. 1 is simplified, but illustrates importantfeatures of the invention. See FIGS. 3-4, 6-8 and 11-12, 14 and 17-18for more detailed schematics.

The chip illustrated in FIG. 2 is designed to carry out five sequentialchemical processes—[¹⁸F]fluoride concentration, water evaporation,radiofluorination, solvent exchange, and hydrolytic deprotection.Referring to FIG. 2, an aqueous potassium fluoride solution istransported by pump 103A from a source through flow channel 100, throughopen valve 102A, and into reactor loop 104, which has at least one gaspermeable portion. The source can be, for example, a fluorideconcentration loop. The solution transported into reactor 104 isretained in the reactor because valves 102B-C are closed. After fillingor partially filling the reactor, valve 102A is closed therebyfluidically isolating the reactor. Reactor 104 is heated using a heaterto evaporate solvent (water) from the reactor. Solvent (Water) vaporescapes reactor 104 though gas permeable material from which thechemical reaction circuit is fabricated, at least in part. Whensufficient solvent is evaporated, valve 102B is opened, an acetonitrilesolution of Kryptofix 222 and d-mannose triflate is transported from asource through flow channel 106 into the reactor 104, and valve 102B isclosed to fluidically isolate the reactor. The ¹⁸F-fluoride andd-mannose triflate react to produce ¹⁸F-FTAG while the reactants arebeing introduced into reactor 104 and/or after valve 102B is closed.Reactor 104 is again heated using the heater to evaporate solvent(acetonitrile) from the reactor though the gas permeable material fromwhich the chemical reaction circuit is, at least in part, composed.After evaporating sufficient acetonitrile, valve 102D is opened and anaqueous HCl solution is transported from a source through flow channel108 and through valve 102D into reactor 104. Valve 102D is then closedto fluidically isolate the reactor 104. The introduction of HCl resultsin hydrolysis of ¹⁸F-FTAG to produce [¹⁸F]FDG, while the reactants arebeing introduced into reactor 104 is being filled and/or after valve102D is closed. Valves 102C and 102E are then opened and water isintroduced into the reactor 104 via flow channel 107, forcing thesolution containing the reaction product through open valve 102E andflow channel 109 to a reservoir or other component of the system.Distribution manifolds (described below) also can be used forintroducing solutions into the reactor.

The synthesis of [¹⁸F]FDG using a device of the type in FIG. 2 and inthe Examples, illustrates a multistep chemical synthesis involvingremoving and exchanging solvents specific to individual synthetic stepsand isolating distinct regions on the chip for individual chemicalprocesses.

Section 3: Microfluidic Device and System

This section describes exemplary materials and components of CRC chips.

A. Materials and Fabrication of Device

Devices of the invention can be constructed out of any material orcombination of materials from which a reactor and an associated networkof channels and valves can be formed. Materials from which a chip can befabricated include, without limitation, elastomers, silicon, glass,metal, polymer, ceramic, inorganic materials, and/or combinations ofthese materials.

The methods used in fabrication of a CRC device will vary with thematerials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491496; and Mastrangeloet al., 1989, Vacuum-Sealed Silicon Micromachined Incandescent LightSource, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506.

In preferred embodiments, the device is fabricated using elastomericmaterials. Fabrication methods using elastomeric materials will only bebriefly described here, because elastomeric materials, methods offabrication of devices made using such materials, and methods for designof devices and their components have been described in detail (see,e.g., Unger et al., 2000, Science 288:113-16; U.S. Pat. Nos. 6,960,437(Nucleic acid amplification utilizing microfluidic devices); 6,899,137(Microfabricated elastomeric valve and pump systems); 6,767,706(Integrated active flux microfluidic devices and methods); 6,752,922(Microfluidic chromatography); 6,408,878 (Microfabricated elastomericvalve and pump systems); 6,645,432 (Microfluidic systems includingthree-dimensionally arrayed channel networks); U.S. Patent Applicationpublication Nos. 2004/0115838, 20050072946; 20050000900; 20020127736;20020109114; 20040115838; 20030138829; 20020164816; 20020127736; and20020109114; PCT patent publications WO 2005/084191; WO05030822A2; andWO 01/01025; Quake & Scherer, 2000, “From micro to nanofabrication withsoft materials” Science 290: 1536-40; Xia et al., 1998, “Softlithography” Angewandte Chemie-International Edition 37:551-575; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-116; Thorsen et al., 2002,“Microfluidic large-scale integration” Science 298:580-584; Chou et al.,2000, “Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330;Liu et al., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23,” Hong et al,2004, “A nanoliter-scale nucleic acid processor with parallelarchitecture” Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005,“Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rolland et al.,2004, “Solvent-resistant photocurable “liquid Teflon” for microfluidicdevice fabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; Terry etal., 1979, A Gas Chromatography Air Analyzer Fabricated on a SiliconWafer, IEEE Trans. on Electron Devices, v. ED-26, pp. 1880-1886; Berg etal., 1994, Micro Total Analysis Systems, New York, Kluwer; Webster etal., 1996, Monolithic Capillary Gel Electrophoresis Stage with On-ChipDetector in International Conference On Micro Electromechanical Systems,MEMS 96, pp. 491496; and Mastrangelo et al., 1989, Vacuum-Sealed SiliconMicromachined Incandescent Light Source, in Intl. Electron DevicesMeeting, IDEM 89, pp. 503-506; and other references cited herein andfound in the scientific and patent literature.

Elastomeric Materials

Elastomers in general are polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature. SeeAllcock et al., Contemporary Polymer Chemistry, 2nd Ed. Elastomericmaterials exhibit elastic properties because the polymer chains readilyundergo torsional motion to permit uncoiling of the backbone chains inresponse to a force, with the backbone chains recoiling to assume theprior shape in the absence of the force. In general, elastomers deformwhen force is applied, but then return to their original shape when theforce is removed. The elasticity exhibited by elastomeric materials maybe characterized by a Young's modulus. Elastomeric materials having aYoung's modulus of between about 1 Pa-1 TPa, more preferably betweenabout 10 Pa-100 GPa, more preferably between about 20 Pa-1 GPa, morepreferably between about 50 Pa-10 MPa, and more preferably between about100 Pa-1 MPa are useful in accordance with the present invention,although elastomeric materials having a Young's modulus outside of theseranges could also be utilized depending upon the needs of a particularapplication.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake the devices of the invention. Common elastomeric polymers includeperfluoropolyethers, polyisoprene, polybutadiene, polychloroprene,polyisobutylene, poly(styrene-butadiene-styrene), polyurethanes, andsilicones, for example, or poly(bis(fluoroalkoxy)phosphazene) (PNF,Eypel-F), poly(carborane-siloxanes) (Dexsil),poly(acrylonitrile-butadiene) (nitrile rubber), poly(1-butene),poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers (Kel-F),poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidenefluoride-hexafluoropropylene) copolymer (Viton), elastomericcompositions of polyvinylchloride (PVC), polysulfone, polycarbonate,polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon),polydimethylsiloxane, polydimethylsiloxane copolymer, and aliphaticurethane diacrylate. For illustration, a brief description of the mostcommon classes of elastomers is presented here:

Silicones: Silicone polymers have great structural variety, and a largenumber of commercially available formulations. In an exemplary aspect ofthe present invention, the present systems are fabricated from anelastomeric polymer such as GE RTV 615 (formulation), a vinyl-silanecrosslinked (type) silicone elastomer (family). The vinyl-to-(Si—H)crosslinking of RTV 615 allows both heterogeneous multilayer softlithography and photoresist encapsulation. However, this is only one ofseveral crosslinking methods used in silicone polymer chemistry andsuitable for use in the present invention. In one embodiment, thesilicone polymer is polydimethylsiloxane (PDMS).

Perfluoropolyethers: Functionalized photocurable perfluoropolyether(PFPE) is particularly useful as a material for fabricatingsolvent-resistant microfluidic devices for use with certain organicsolvents. These PFPEs have material properties and fabricationcapabilities similar to PDMS but with compatibility with a broader rangeof solvents. See, e.g., PCT Patent Publications WO 2005030822 and WO2005084191 and Rolland et al., 2004, “Solvent-resistant photocurable“liquid Teflon” for microfluidic device fabrication” J. Amer. Chem. Soc.126:2322-2323.

Polyisoprene, polybutadiene, polychloroprene: Polyisoprene,polybutadiene, and polychloroprene are all polymerized from dienemonomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). Homogeneous multilayersoft lithography would involve incomplete vulcanization of the layers tobe bonded and photoresist encapsulation would be possible by a similarmechanism.

Polyisobutylene: Pure Polyisobutylene has no double bonds, but iscrosslinked to use as an elastomer by including a small amount (˜1%) ofisoprene in the polymerization. The isoprene monomers give pendantdouble bonds on the polyisobutylene backbone, which may then bevulcanized as above.

Poly(styrene-butadiene-styrene): Poly(styrene-butadiene-styrene) isproduced by living anionic polymerization (that is, there is no naturalchain-terminating step in the reaction), so “live” polymer ends canexist in the cured polymer. This makes it a natural candidate for thepresent photoresist encapsulation system (where there will be plenty ofunreacted monomer in the liquid layer poured on top of the cured layer).Incomplete curing would allow homogeneous multilayer soft lithography (Ato A bonding). The chemistry also facilitates making one layer withextra butadiene (“A”) and coupling agent and the other layer (“B”) witha butadiene deficit (for heterogeneous multilayer soft lithography). SBSis a “thermoset elastomer”, meaning that above a certain temperature itmelts and becomes plastic (as opposed to elastic); reducing thetemperature yields the elastomer again. Thus, layers can be bondedtogether by heating.

Polyurethanes: Polyurethanes are produced from di-isocyanates (A-A) anddi-alcohols or di-amines (B-B); since there are a large variety ofdi-isocyanates and di-alcohols/amines, the number of different types ofpolyurethanes is huge. The A vs. B nature of the polymers, however,would make them useful for heterogeneous multilayer soft lithographyjust as RTV 615 is: by using excess A-A in one layer and excess B-B inthe other layer.

The selection of materials (whether elastomeric or non-elastomeric) willtake into account the need for particular material properties and willdepend on a variety of factors including: ease of manufacture, thenature of the chemical synthesis, solvent resistance and temperaturestability. For example, fluidic circuits fabricated from PDMS will notbe compatible with all organic solvents (see, e.g., Lee et al., 2003,Anal. Chem. 75:6544-54). This issue can be addressed by the use ofchemically resistant elastomers in place of PDMS in at least someregions of the device. For example, perfluoropolyether (PFPE) can beused (see Rolland et al., 2004, “Solvent-resistant photocurable “liquidTeflon” for microfluidic device fabrication” J. Amer. Chem. Soc.126:2322-23, and citations herein above). Alternatively, the elastomer(e.g., PDMS) surface can be chemically modified to increasecompatibility with organic solvents and improve function Methods andreagents for such modification include those described in US2004/0115838 [para. 0293] et seq.; copolymers of tetrafluoroethylene,perfluoromethylvinylether (also called TFE-perfluorovinylether polymers)such as Chemraz (Greene-Tweed, 10% solution) diluted 1:1 in low boilingpoint perfluorocarbon liquid, e.g. Flourinert from 3M), Kalrez (DuPont), Chemtex (Utex Industries), and fluorocarbon polymers (FKM, e.g.poly(tetrafluoro-co-hexafluoropropylene) such as Cytop coating(poly(perfluoro (alkenyl vinyl ether) from Bellex International Corp.and Novec EGC-1700 coating (fluoroaliphatic polymer) from 3M which canbe applied by flushing the solutions though channels (e.g., 3×40microliters at 25 psi, at 1 min intervals). In addition, many chemicalreactions can be carried out in a variety of solvents. Reaction seriesto be carried out in a chip made using particular materials can bedesigned to use solvents that are compatible with the materials over theperiod of time necessary to complete the reaction.

For devices made using multilayer soft lithography (in which layers ofelastomer are cured separately and then bonded together) anotherimportant consideration for fabrication is the ability to bond multiplelayers of elastomers together. This scheme requires that cured layerspossess sufficient reactivity to bond together. Either the layers may beof the same type, and are capable of bonding to themselves, or they maybe of two different types, and are capable of bonding to each other.Other possibilities include the use an adhesive between layers, the useof thermoset elastomers, and use of composite structures.

Elastomeric Fabrication Methods

Methods of fabrication of complex microfluidic circuits usingelastomeric are known and are described in Unger et al., 2000, Science288:113-116; Quake & Scherer, 2000, “From micro to nanofabrication withsoft materials” Science 290: 1536-40; Xia et al., 1998, “Softlithography” Angewandte Chemie-International Edition 37:551-575; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-116; Thorsen et al., 2002,“Microfluidic large-scale integration” Science 298:580-584; Chou et al.,2000, “Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330;Liu et al., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23,” and otherreferences cited herein and known in the art.

Microfluidic devices are generally constructed utilizing single andmultilayer soft lithography (MSL) techniques and/or sacrificial-layerencapsulation methods. The basic MSL approach involves casting a seriesof elastomeric layers on a micro-machined mold, removing the layers fromthe mold and then fusing the layers together. In the sacrificial-layerencapsulation approach, patterns of photoresist are deposited wherever achannel is desired. One exemplary method for fabricating elastomericdevices is briefly described below.

In brief, one method for fabricating elastomeric devices involvefabricating mother molds for top layers (the elastomeric layer with thecontrol channels and reactors, the elastomeric layer with the flowchannels) on silicon wafers by photolithography with photoresist(Shipley SJR 5740). Channel heights can be controlled precisely by thespin coating rate. Photoresist channels are formed by exposing thephotoresist to UV light followed by development. Heat reflow process andprotection treatment is typically achieved as described by Unger et al.supra. A mixed two-part-silicone elastomer (GE RTV 615) is then spuninto the bottom mold and poured onto the top mold, respectively. Spincoating can be utilized to control the thickness of bottom polymericfluid layer. The partially cured top layer is peeled off from its moldafter baking in the oven at 80° C. for 25 minutes, aligned and assembledwith the bottom layer. A 1.5-hour final bake at 80° C. is used to bindthese two layers irreversibly. Once peeled off from the bottom siliconmother mold, this RTV device is typically treated with HCL (0.1N, 30 minat 80° C.). This treatment acts to cleave some of the Si—O—Si bonds,thereby exposing hydroxy groups that make the channels more hydrophilic.

The device can then optionally be hermetically sealed to a support. Thesupport can be manufactured of essentially any material, although thesurface should be flat to ensure a good seal, as the seal formed isprimarily due to adhesive forces. Examples of suitable supports includeglass, plastics and the like.

The devices formed according to the foregoing method result in thesubstrate (e.g., glass slide) forming one wall of the flow channel.Alternatively, the device once removed from the mother mold is sealed toa thin elastomeric membrane such that the flow channel is totallyenclosed in elastomeric material. The resulting elastomeric device canthen optionally be joined to a substrate support.

Access to the fluidic channels is achieved by punching holes through thebulk material, and the devices are readily bonded to glass or siliconsubstrates. Large arrays of active components, such as channels,reactors, valves and pumps, can be created by stacking multiple,individually fabricated layers.

Composite Structures

Diverse materials can be used in fabrication of the chip and reactor.Devices, and in particular, reactors, can be fabricated fromcombinations of materials. For example, in some embodiments the wallsand ceiling of a reactor are elastomeric and the floor of the reactor isformed from an underlying nonelastomeric substrate (e.g., glass), whilein other embodiments, both the walls and floors of the reactor areconstructed from a nonelastomeric material, and only the ceiling of thereactor is constructed from elastomer. These chips and reactors aresometimes referred to as “composite structures.” See, e.g., US20020127736. A variety of approaches can be employed to seal theelastomeric and nonelastomeric components of a device, some of which aredescribed in U.S. Pat. No. 6,719,868 and US 20020127736, ¶¶0227 et seq.

B. Basic Device Components: Flow Channels, Reactors, Valves FlowChannels

The term “flow channel” refers to a microfluidic channel through which asolution can flow. The dimensions of flow channels can vary widely buttypically include at least one cross-sectional dimension (e.g., height,width, or diameter) less than 1 mm, preferably less than 0.5 mm, andoften less than 0.3 mm. Flow channels often have at least onecross-sectional dimension in the range of 0.05 to 1000 microns, morepreferably 0.2 to 500 microns, and more preferably 10 to 250 microns.The channel may have any suitable cross-sectional shape that allows forfluid transport, for example, a square channel, a circular channel, arounded channel, a rectangular channel, etc. In an exemplary aspect,flow channels are rectangular and have widths of about in the range of0.05 to 1000 microns, more preferably 0.2 to 500 microns, and morepreferably 10 to 250 microns. In an exemplary aspect, flow channels havedepths of 0.01 to 1000 microns, more preferably 0.05 to 500 microns,more preferably 0.2 to 250 microns, and more preferably 1 to 100microns. In an exemplary aspect, flow channels have width-to-depthratios of about 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1, and often about10:1. As shown in FIG. 3, flow channels in elastomeric devices may havea curved or elliptical face that allows the deflected elastomericmembrane is fully compliant to the round-profile fluidic channel andallowing complete closure of monolithic valves (except at the positionsof sieve valves, as discussed below). In one embodiment the flow channeldimensions are 250-300 microns by 45 microns. Although certain preferredembodiments have been described, the flow channels of the invention arenot limited to the dimensions above.

At least some flow channels of a chip of the invention are in fluidiccommunication with a reactor (described below). In one embodiment, theflow channel is configured as a distribution manifold, and referenceherein to a flow channel in fluidic communication with a reactor isintended to include distribution manifolds unless otherwise indicated orclear from context. A distribution manifold is a configuration of a flowchannel that serves divide flow into several parts, with the parts beingintroduced through different ports into the same reactor (see, e.g.,FIGS. 11 and 12). In a preferred embodiment, the solution is introducedequally and simultaneously through the ports. In one embodiment, amanifold for introduction of a solution to a reactor, particularly a“coin-shaped” reactor, is fashioned generally as shown in FIGS. 11 and12. The six-channel manifold allows a solution to enter the chamber from6 directions simultaneously which leads to faster mixing and shorterreaction times. Simultaneous introduction of liquid is accomplished byhaving equal path lengths in the channel work from the origin of themanifold to each opening to the chamber. It is also facilitated byhaving one valve at the source of the manifold and a second set ofvalves at the entrances of the channels to the chamber—this allows themanifold to be filled first, prior to releasing the fluid into thereactor. In alternative embodiments the distribution manifold has 4-10channels equidistant from the first splitting point.

Reactors

In one aspect, the invention provides a method for carrying out achemical reaction using a microfluidic device that has at least onereactor (also referred to as a “reaction chamber”). In some embodiments,the device has multiple reactors, which may be configured serially, inparallel, or otherwise, for carrying out multiple and/or parallelreaction series.

In general, a reactor is characterized by the following threeproperties:

1. The Reactor is Configured to Fluidically Communicate with at LeastOne Flow Channel

The reactor is configured to fluidically communicate with at least oneflow channel. Typically the reactor is in fluidic communication withmore than one flow channel such as at least two, at least three, atleast four, at least five, at least six, or more than six different flowchannels. For illustration, a reactor may be in fluidic communication orconfigured to fluidically communicate with 1 to 20 flow channels, 1 to10 flow channels, 2 to 10 flow channels, 3 to 10 flow channels, 4 to 10flow channels or 5 to 10 flow channels. As discussed above, in someembodiments one or more flow channels are configured as a distributionmanifold(s). In one embodiment the reactor is in fluidic communicationwith at least one distribution manifold (considered a single channel forthe purposes of enumeration).

2. The Reactor is Configured to be Fluidically Isolated

The reactor is configured so that is can be fluidically isolated.Typically this is accomplished by closing (actuating) valves so as toprevent liquid flow from a flow channel into the reactor or from thereactor to a flow channel. Thus, when a reactor is fluidically isolatedliquid cannot, to any significant degree, flow out of the reactor.

3. The Reactor is Permeable to a Gas But Substantially Impermeable tothe Liquid Corresponding to the Gas and to Reactants and ProductsDissolved in the Liquid

The reactor is adapted for solvent exchange. That is, at least a portionof the reactor wall is selectively permeable so that when the reactor isfluidically isolated (and thus any liquid in the reactor is confined tothe chamber) vapor can escape by passing through the reactor wall. Thus,a solvent (e.g., acetonitrile) in liquid phase is contained in afluidically isolated reactor but escapes the fluidically isolatedreactor when converted to gas phase. A liquid solvent is converted tothe gas phase by evaporation and/or by heating the liquid and/orreducing the ambient pressure. In one embodiment, the liquid in thereactor is heated to or above its normal (atmospheric) boiling point. Inone embodiment, the reactor wall is essentially impermeable to liquidwater (thus water does not leak through the chamber wall and out of thedevice) but is permeable to water vapor. Exemplary solvents to which thereactor wall (or gas permeable portion thereof) is selectively permeablecan include one or more of water; acetic acid; acetone; acetonitrile;benzene; 1-butanol; 2-butanol; 2-butanone; t-butyl alcohol; carbontetrachloride; chlorobenzene; chloroform; cyclohexane;1,2-dichloroethane; diethyl ether; diethylene glycol; diglyme(diethylene glycol dimethyl ether); 1,2-dimethoxy-ethane (glyme, DME);dimethyl-formamide (DMF); dimethyl sulfoxide (DMSO); dioxane; ethanol;ethyl acetate; ethylene glycol; glycerin; heptane;hexamethylphosphoramide (HMPA); hexamethylphosphorous triamide (HMPT);hexane; methanol; methyl t-butyl; ether (MTBE); methylene chloride;N-methyl-2-pyrrolidinone (NMP); nitromethane; pentane; Petroleum ether(ligroine); 1-propanol; 2-propanol; pyridine; tetrahydrofuran (THF);toluene; triethyl amine; and xylene, and combinations thereof. It willbe understood that the reactor wall may be permeable to gas mixturessuch as, for example, the mixture created by evaporation of anazeotrope, such as the azeotrope of water and acetonitrile.

In one embodiment, the reactor is formed, at least in part, by a gaspermeable elastomeric material, such as an elastomer described above.Certain elastomers, such as PDMS and PFPE, in particular arecharacterized by outstanding gas permeability to certain solvents.

Other materials (not necessarily considered elastomeric) may also beused. For example, the permeable material may include a polymer (e.g., asingle polymer type, a co-polymer, a polymer blend, a polymerderivative, etc.) including polyfluoroorganic materials such aspolytetrafluoroethylenes and amorphous fluoropolymers; polystyrenes;polysulfones; polycarbonates; acrylics (e.g., polymethyl acrylate andpolymethyl methacrylate); polyethylenes (e.g., high, low, ultra low, andlinear low-density polyethylenes); polyvinylchlorides;poly(4-methylpentene-1) (“PMP”); poly(4-methylhexene-1),poly(4-methylheptene-1); poly(4-methyloctene-1), andpoly(1-trimethlsilyl-1-pro-pyne).

In some embodiments, the entire interior surface of the reactor (e.g.,“floor,” “ceiling,” and “walls”) is composed of a gas permeablematerial. This may be the case when the device is fabricated from PDMS,for example. In some embodiments, at least a portion of the interiorsurface of the reactor is defined by a material that is not a gaspermeable material (e.g., silicon, glass or metal). For example, for areactor of the device can have a floor and walls fabricated from glass,and have a “ceiling” fabricated from PDMS (see, e.g., description ofcomposite structures above).

In some embodiments, the device is constructed so that the distance fromthe interior surface of the reactor to the exterior of the device, or toa vent to the exterior such as the lumen of a vent channel is less than1000 microns (i.e., the thickness of the gas-permeable material thevapor must pass through to reach the topological exterior of thedevice). In some embodiments, the thickness of the gas-permeablematerial is from 1 to 1000 microns, sometimes from 1 to 1000 microns,often from 50 to 500 microns, and most often from 50 to 200 microns,e.g., 100 microns. It will be appreciated that the optimal orappropriate thickness will vary depending on the material and intendeduse of the reactor. In some embodiments, the device has channels,referred to as “vent channels” positioned to accelerate or facilitatewithdrawal of gas from the reactor. Vent channels are described below.

Alternatively the solvent can be evaporated out of the reactor and intoan elastomeric or other gas-permeable material and remain in thematerial (i.e., without any substantial amount of the gas reaching theexterior).

In addition to the properties above, in certain embodiments, a reactormay have one or more of the following properties (related to reactorsize, number, shape, and relationship to a chromatography column):

4. The Liquid Capacity of a Reactor May be Large

The volume (or liquid capacity) can vary widely from the nanoliter tomicroliter range. In certain embodiments the reactor capacity is lessthan 1 microliter (e.g., 1 nL to 1000 nL, often 100 nL to 500 nL). Incertain embodiments, the volume or liquid capacity of the reactor isgreater than 1 microliter, sometimes greater than 5 microliters, andsometimes greater than 10 microliters. In certain embodiments, thevolume of the reactor is from 1 to 20 microliters, 2 to 20 microliters,5 to 20 microliters, or 10 to 20 microliters. In certain embodiments,the volume of the reactor is from 1 to 10 microliters, 2 to 10microliters, 5 to 10 microliters, or 7 to 10 microliters.

5. The Reactor May have a Variety of Geometries

The reactor may have a variety of geometries or interior shapes. Theselection of reactor shape will vary with the intended use of the devicesuch as, in some cases, the volume of reactants or solutions to beintroduced into the reactor, the quantity of product desired, and otherfactors. For example, if the reactant is eluted from a column andtransported in its entirely to a reactor, the reactor would usually havea volume equal to the elution volume (alternatively, one or more roundsof solvent removal could be used to reduce the volume). Preferably theshape and dimensions of the reactor are selected to allow efficientmixing of solutions introduced into the reactor. Exemplary shapesinclude tubular, spherical, cylindrical, polyhedral (e.g., ahexahedron), coin-shaped, box-like, bar-bell shaped, and others. In oneembodiment the reactor has an irregular shape. In some embodiments, areactor chamber may include baffles or other structures to increasemixing efficiency.

In one embodiment, the reactor has the form of a flow channel (or loopchannel) that can be isolated from other channels to form a closed paththrough which fluid can circulate (see Example 1, below). Loop channelsare described in U.S. Pat. No. 6,767,706. Typically such a reactor hasdimensions that fall within the ranges provided above for flow channels(e.g., 200 microns by 45 microns) although larger dimensions can be usedto accommodate larger volume reactions. A closed path means that thechannel can be temporarily isolated from other parts of the chip, forexample by closing valves in any channels which lead into or out of theloop, and that a liquid can then circulate through the path. Pumps suchas peristaltic pumps, can be used to circulate liquid. Alternatively,other mechanisms can be used for circulation and/or mixing in theisolated reactor.

In one embodiment, the reactor has the form of a circular loop channel.In one embodiment, the reactor has the form of a loop channel other thana circular channel. See, for example, FIG. 20 of U.S. Pat. No. 6,767,706for a description of a closed loop channel in which a solution may becirculated or two solutions circulated and mixed.

In some embodiments the reactor has a shape other than a loop channel.In some embodiments the dimensions of the reactor are such that theheight, width and length or height and diameter, or the like, vary by nomore than a factor of 50 (i.e., the longest dimension is no more than50-fold as much as the shortest dimension). In other embodiments thedimensions of the reactor vary by no more than a factor of 40, no morethan 30, no more than 20, no more than 10, no more than 5 or no morethan 2.

In one embodiment the reactor is “coin-shaped.” That is, roughly acylinder with a high diameter to height ratio, usually greater than 5,usually greater than 10, often greater than 15, and sometimes 20 orgreater. An exemplary reactor has a height of from 25 to 1,000micrometers and a diameter of from 1,000 to 20,000 micrometers. Anexemplary reactor has dimensions of 250 micrometers (height) by 5000micrometers (diameter). In one embodiment, the reactor has the shape ofa wide and short cylinder (coin-shaped, 250 um in height and 5 to 7 mmdiameters). See FIGS. 11 and 12.

In other embodiments the reactor is “box-like.” That is, having arectangular floor and ceiling and a height dimension significantlysmaller than the other dimensions (e.g., a high width to height ratio,usually greater than 5, usually greater than 10, often greater than 15,and sometimes 20 or greater). In related embodiments, the reactor has afloor and/or ceiling with the shape of any regular or irregularparallelogram. In other embodiments, the reactor has a floor and/orceiling with an irregular shape. In other embodiment the interior shapeof the reactor is roughly spherical or roughly cubical, or has adifferent aspect ratio than described above.

In certain embodiments, the reactor does not have the form of a circularflow channel and does not form a closed path. For example, a coin-shapedreactor does not have the form of a circular flow channel and does notform a closed path. In certain embodiments, a reactor has the form of acircular flow channel but has cross-sectional dimensions that do notfall within the ranges provided above for flow channels (<1 mm). Incertain embodiments, the interior of the reactor does not have the shapeof a tube. In certain embodiments, the reactor does not have the shapeof a circular loop channel. In certain embodiments, the reactor does nothave the shape of a noncircular loop channel. In certain embodiments,the interior of the reactor does not have the shape of a polyhedron. Incertain embodiments, the reactor is not coin-shaped. In certainembodiments, the reactor is not cylindrical. Exemplary shapes includetubular, spherical, cylindrical, polyhedral (e.g., a hexahedron),coin-shaped, box-like, and others.

6. The Device May have a Small Number of Reactors

In certain embodiments, the device has a single reactor. In otherembodiments, the device has 2-5 reactors. In other embodiments, thedevice has 2-10 reactors, or 2-50 reactors. In other embodiments thedevice may have up to 10,000 reactors.

7. The Reactor is Configured to Fluidically Communicate with aMicrofluidic Separation Column or Concentration Loop Including aMicrofluidic Separation Column

In certain embodiments the reactor is configured to fluidicallycommunicate with a microfluidic separation column. That is, an eluatefrom the column can be transported from the column to the reactor, orfrom the reactor to a column. The column can be on the chip (forexample, formed in a flow channel or similarly integral to the chip) orexternal to the chip. In one embodiment the column is on the chip.Exemplary on-chip microfluidic separation columns (“columns”) aredescribed below. Examples of off-chip columns include any columnssuitable for chromatography of small volumes. An off chip column devicemay have the fluidic inputs and outputs and controlling valve functionsperformed by the chip or microfluidic device.

In some embodiments, the device comprises a concentration loop. Aconcentration loop includes an on-chip separation column (through whicha solution can pass), a column inlet, a column outlet, and a flowchannel or channels that connect the outlet to the inlet, and sufficientvalves such that when valves between the loop and other flow channelsare closed, flow channel(s) and the separation column(s) define a closedpath through which fluid can circulate. In a preferred embodiment, theconcentration loop includes a pump, preferably a peristaltic pump,capable of moving a solution through the closed path such that thesolution flows through the column multiple times. See FIGS. 2 and 3 andaccompanying text for illustrations of such a column and closed path. Inone embodiment, the concentration loop has a generally rectangularconfiguration. As describe in Example 1, the concentration loopconfiguration allows a solution to be circulated multiple times thoughthe column to ensure efficient binding of a compound in the solution tothe stationary phase of the column and, in a similar manner, allows anelution solution to flow multiple times though the column to ensure ahigh degree of elution of reactant or product from the column.

In some embodiments, the solution containing the reaction product istransported from a reactor to a column (e.g., an on-chip column) for,for example, purification or concentration of the product. The eluatefrom the column can be transported back to the reactor for furthermodification of the produce or solvent system. More often the eluatefrom the column can be transported to a different reactor. Alternativelythe eluate can be transported to another chip component or to anoff-chip component (including, for example, a collection vial).

Valves

Valves of the microfluidic device can be selectively actuated (and/orare one-way valves) to regulate flow in and between channels, reactors,and other chip components. Valves of the device serve to block flowwithin a flow channel, from a flow channel into a reservoir or reactor,from a reservoir or reactor to a flow channel, or at other sites inwhich liquid flows.

Valves of various types are known in the art, including micromechanicalvalves, elastomeric valves, solid-state microvalves, and others. See,e.g., Felton, 2003, The New Generation of Microvalves” AnalyticalChemistry 429-432. Two common approaches to fabrication ofmicroelectromechanical (MEMS) structures such as pumps and valves aresilicon-based bulk micro-machining (which is a subtractive fabricationmethod whereby single crystal silicon is lithographically patterned andthen etched to form three-dimensional structures), and surfacemicro-machining (which is an additive method where layers ofsemiconductor-type materials such as polysilicon, silicon nitride,silicon dioxide, and various metals are sequentially added and patternedto make three-dimensional structures).

In one embodiment, the valve is a monolithic valve. In a preferredembodiment the valve is a pressure-actuated “elastomeric valve.” Apressure-actuated elastomeric valve consists of a configuration in whichtwo microchannels are separated by an elastomeric segment that can bedeflected into or retracted from one of the channels (e.g., a flowchannel) in response to an actuation force applied to the other channel(e.g., a control channel). Examples of elastomeric valves includeupwardly-deflecting valves (see, e.g., US 20050072946), downwardlydeflecting valves (see, e.g., U.S. Pat. No. 6,408,878), side actuatedvalves (see, e.g., US 20020127736, e.g., paragraphs 0215-0219],normally-closed valves (see, e.g., U.S. Pat. No. 6,408,878 B2 and U.S.Pat. No. 6,899,137) and others. In some embodiments a device can have acombination of valves (e.g., upwardly and downwardly deflecting valves).Valves can be actuated by injecting gases (e.g., air, nitrogen, andargon), liquids (e.g., water, silicon oils and other oils), solutionscontaining salts and/or polymers (including but not limited topolyethylene glycol, glycerol and carbohydrates) and the like into thecontrol channel. Some valves can be actuated by applying a vacuum to thecontrol channel.

In addition to elastomeric valves actuated by pressure-based actuationsystems, monolithic valves with an elastomeric component andelectrostatic, magnetic, electrolytic and electrokinetic actuationsystems may be used. See, e.g., US 20020109114; US 20020127736, e.g.,¶¶0168-0176; and U.S. Pat. No. 6,767,706 B2 e.g., § 6.3. One-way valveshave also been described (see, e.g., Adams et al., 2005, J. Micromech.Microeng. 15:1517-21; and references 6-12 therein)

In some embodiments, pairs of valves are used, with one acting as a“back-up valve” or “double valves.” See, e.g., FIG. 11. Back-up valvesare used to confine reaction mixtures in the event the primary valvefails (e.g., due to the relatively higher vapor pressure the valves maybe subjected to during the solvent exchange process). Bursts of highpressure may be generated inside the reaction chamber that are strongenough to push the valves open at least briefly (e.g., for a fraction ofa second). In such an event, if there is back pressure behind the closedvalve, such valve may close back down after being briefly opened withoutloss of pressure inside the reactor. If there is a much lower pressurebehind the valve, some liquid may escape from the chamber, in turnpushing the valve open further. The back pressure behind the valvessurrounding the reaction chamber is achieved by having a second set ofvalves a short distance from the first ones.

C. Other Device Components

Vent Channels

In one embodiment, the device has channels, referred to as “ventchannels” positioned to accelerate or facilitate withdrawal of gas fromthe reactor during solvent exchange or reactor filling (e.g., dead-endor blind filling). A vent channel system comprises channels separatedfrom a reactor by a thin gas permeable (e.g., elastomeric) membrane. Thevent channels typically lie over or under a reactor (e.g., in a ventlayer or control layer). Vapor can be drawn out of the reactor, passthrough an intervening gas permeable material (such as an elastomer),and enter the vent channels(s). Vapor can diffuse into the vent channelor removal can be accelerated by reducing the pressure in the ventchannel relative to the reactor chamber. This reduction can be achieved,for example, by flowing gas through the vent channel(s) or drawing avacuum through the channel(s), as described below, or by any othermethod that reduces vent channel pressure. Thus, vent channels can beused for accelerating evaporation to concentrate a solute and reduce thevolume of a liquid in a chamber. This mechanism for accelerating solventevaporation is particularly valuable when a large (microliter) volumereactor is used.

The dimensions of vent channels can vary widely. In an exemplary aspect,vent channels have at least one cross-sectional dimension in the rangeof 0.05 to 1000 microns, often 50 to 500 microns, and most often 100 to400 microns. In some embodiments, the channel height is not more thanabout 500 microns or less than about 20 microns (in some embodiments,not more than about 250 microns or less than about 50 microns) and thechannel width is not more than 5000 microns or less than 20 microns). Inone embodiment, vent channels have rectangular cross-sectionaldimensions of about 250 microns×250 microns. In some embodiments, ventchannels preferably have width-to-depth ratios of about 1:10 to 100:1,such as between about 2:1 and 1:2, and sometimes about 1:1. Inembodiments in which a vacuum is applied to a vent channel dimensionsmay be selected to avoid collapse of the channel under vacuum (e.g.,higher height:width ratios). However, the vent channels are not limitedto these particular dimensions or proportions.

As noted above, in some embodiments, the lumen of the vent channel(s) isseparated from the interior of the reactor by less than 1000 microns,such as from 10 to 1000 microns, often from 50 to 1000 microns, oftenfrom 50 to 500 microns, and most often from 50 to 200 microns, e.g., 100microns. In one embodiment, a vent is placed above the chamberconsisting of a radiator of 250×250 micron channels separated from thechamber by a 100 micron membrane (gas-permeable). See, e.g., FIG. 11.

With reference to an elastomeric or partially elastomeric device, asystem of vent channel can lie in an elastomer layer one side of whichconstitutes a portion of the interior surface of the reactor. Forexample, in a “wholly” elastomeric device the vent channels may lie inthe elastomer layer above or below the flow channel layer (and, fordevices with control channels, on the side of the flow layer oppositethe control channel layer or in the control channel layer). Ventchannels may also be incorporated into the flow channel layer. In someembodiments, providing vent channels above the reaction chamber is theoptimal arrangement. However, it is generally easier to fabricate an MSLchip with the vent below the chamber (e.g., as part of the controllayer). Dead-end filling rates were similar in both arrangements. It wasobserved that solvent evaporation, while facilitated significantly byboth kinds of vents, was less efficient with the bottom vent location inpart because in this arrangement, vapors condensed on the ceiling of thereaction chamber.

In one embodiment, vent channels can function passively (by providing arelatively short path from the isolated reactor to the topologicalexterior of the device). The devise shown in FIG. 14, has six ventchannels (“vent lines”) in communication with the atmosphere. The ventchannels in FIG. 14 are situated in the control layer. In otherembodiments, the use of a stream of gas, or, preferably application of avacuum, can significantly accelerate the rate of solvent evaporation.Thus, in one embodiment, the vent channel(s) are optionally configuredwith a vacuum pump (or equivalent device) to draw a vacuum in the ventchannels and withdraw gas from the reactor. The vacuum pump can be oncontinuously or actuated while certain chemical processes are underway.In an alternative embodiment, dry gas (e.g., air or N₂) can be flowedthrough the channel to remove vapor from a reactor.

Application of vacuum to the vent channel allows fast removal of gasfrom the chamber when the latter needs to be filled with fluid. Alsoduring evaporation, it allows removal of solvent vapors. As a result,not only it can it speed up the evaporation, but it also reduces thevapor pressure, which allows some solvents to be removed at lowertemperatures. Use of a vent channel system can also reduce pressure onclosed valves during the evaporation steps.

A number of vent configurations are possible. In a preferred embodiment,the vent has two open ends to facilitate flushing the vapors (which maycondense inside) out of the chip (for example, by applying N₂ gas). SeeFIG. 20.

A vent system also can be configured so as to not accelerate evaporationat certain times in a reaction or series of reactions when suchacceleration is not desired due to reaction kinetics or for otherreasons. The functioning of a vent system can be modulated by ceasinggas flow or turning off a vacuum source, as appropriate. The acceleratedevaporation caused by passive vents can be eliminated or reduced byfilling the vents with an oil or similar fluid.

Usually a vent channel system is localized over a reactor or set ofreactors rather than being, for example, distributed uniformlythroughout the area or footprint of the device. In one embodiment, asubstantial portion of a vent channel overlies a single chamber. In thiscontext, a substantial portion means that at least 10% of the length ofthe vent channel lies over the chamber, preferably at least 20%, andmost preferably at least 30%, and sometimes at least 50% of the lengthof the vent channel lies over the chamber. This is illustrated in FIG.11B in which about 30% of the length of the vent channel (blue) liesover the 5 ul coin-shaped reactor. Equivalently, in one embodiment, notmore than 90%, preferably not more than 80%, most preferably not morethan 70%, and sometimes not more than 50% of the length of the channellies over a region or regions of the device other than a solventexchange chamber. In one embodiment, a substantial portion of a ventchannel overlies a single chamber. In this context, a substantialportion means that at least 10% of the length of the vent channel liesover the chamber, preferably at least 20%, and most preferably at least30%, and sometimes at least 50% of the length of the vent channel liesover the chamber.

In a related embodiment, a device has more than one reactor and asubstantial portion of a vent channel overlies a two or more chambers ofa device. In this context, a substantial portion means that at least 10%of the length of the vent channel lies over the chambers, preferably atleast 20%, and most preferably at least 30%, and sometimes at least 50%of the length of the vent channel lies over the chambers. Equivalently,in one embodiment, not more than 90%, preferably not more than 80%, mostpreferably not more than 70%, and sometimes not more than 50% of thelength of the channel lies over a region or regions of the device otherthan one of the solvent exchange chambers.

Evaporation can be accelerated by any method for introducing adifferential in the chemical potential for the gas molecule so that itis lower outside the chamber than inside, including increasing pressurewithin the reactor, reducing pressure or solvent concentration outsidethe reactor, placing or flowing a solution in which the solvent gas ishighly soluble in a microchannel or chamber separated from the reactorchamber by the gas-permeable (e.g., elastomeric) membrane.

The invention provides methods for rapidly removing solvent (e.g.,water, acetonitrile, alcohols) from a chamber using the methods above.In some embodiments the evaporation is accelerated by heating. In someembodiments heat is not used. For example, a large volume of solvent(e.g., a volume bounded by the range having a lower value of 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 microliters and an upper value of 20, 15, 10 or 5microliters, where the upper value is greater than the lower value) canbe evaporated (i.e., at least 90%, preferably at least 95% of the volumeis evaporated) rapidly (i.e., less than 3 minutes, preferably less than2 minutes, more preferably less than 1 minute, more preferably less than45 seconds, and sometimes less than 30 seconds). In another embodiment asolvent volume between 100 and 1000 nanoliters is rapidly evaporatedfrom the chamber (e.g., preferably less than 1 minute, more preferablyless than 45 seconds, and sometimes less than 30 seconds).

In a different approach, solvent can be removed by adding a precipitantcausing the reactants and/or product to be precipitated. By using asieve valve, partially opened valve, or other filtering feature theprecipitant can be trapped, the solvent eluted and replaced and theprecipitant re-dissolved into solution using a second solvent systemintroduced through any of the valves.

Separation Devices (Chromatography Columns)

In one embodiment, the device includes one or more separation devicesused to separate, purify or concentrate reactants, products or othercompounds. Separation devices may be based on electrophoresis,centrifugation, and other separation methods. In some embodiments theseparation device is a miniaturized chromatographic column (i.e., amicrofluidic separation column) adapted to perform a chromatographicseparation process. One particularly useful separation process is liquidchromatography, which may be used with a wide variety of sample typesand encompasses a number of methods that are used for separating ions ormolecules that are dissolved in or otherwise mixed into a solvent. Asused in this context, “sample” refers to a solution containing aproduct, reactant or other reagent to be concentrated, purified,separated from other components of the solution and/or transferred to adifferent solvent.

In liquid chromatography a liquid “mobile phase” (typically consistingof one or more solvents) carries a sample containing multipleconstituents or species through a separation medium or “stationaryphase.” Stationary phase material typically includes a liquid-permeablemedium such as beads, packed granules (particulate material) or a porousmonolith disposed within a tube or a channel boundary which may bederivatized, bound to or coated with a compound(s) that specificallyinteracts with a compound in solution as it passes through the column.In one embodiment the chromatography column is a microfluidic channelhaving a stationary phase that is bonded to a functional group on theinner surface of the channel.

The mobile phase may be forced through the stationary phase using pumps,voltage-driven electrokinetic flow, or other methods for generating apressure differential. After a sample is applied to the column,components of the sample will migrate according to interactions with thestationary phase and the flow of such components are retarded to varyingdegrees. Individual sample components (e.g., reactants or products) mayreside for some time in the stationary phase until conditions (e.g., achange in solvent) permit a component to emerge from the column with themobile phase.

The columns of the present invention can be selected to retain thereactant/product when the sample is applied to the column and release itunder certain conditions such as, typically, application of an elutionsolvent or elution solution. Alternatively, the reactant/product maypass rapidly though the column when the sample is applied, and other(undesired) components of the sample may be retained.

Exemplary separation devices are described in US 2002/0164816; and US2004/0115838, e.g., para 0327-0333, and U.S. Pat. No. 6,752,922(describing microfabricated chromatography column configured in a rotarychannel). Examples of chromatographic separation material can include abead material (e.g., cross-lined agarose or dextran beads,functionalized silica, polymer-coated silica, or porous silicaparticles, resins such as copolymers of styrene and divinylbenzen, anddivinylbenzene and acrylic or methacrylic acid, metal and othermaterials) which may be derivatized, bound to or coated with acompound(s) that specifically interacts with a compound in solution asit passes through the column. For example and without limitation,chromatographic separation material can be adapted for many types ofchromatography including gel filtration, anion exchange, cationexchange, hydrophobic interaction, size exclusion, reverse phase, metalion affinity chromatography, IMAC, immunoaffinity chromatography, andadsorption chromatography. For example and not limitationchromatographic separation material that can be used in the columnmodule can be ion exchange resins (e.g., anion-exchange resins,cation-exchange resins), affinity chromatography resins, size exclusionchromatography resins, and others. Examples of useful resins include HEIX8 (BioRad Corp.) and Source 15Q (Amersham Biosciences).

Exemplary On-Chip (Integral) Columns

In one embodiment, a column is constructed by trapping stationary phasebeads (e.g., ion exchange beads) in a fluid channel isolated withpartially closed valves (see, Hong et al, 2004, “A nanoliter-scalenucleic acid processor with parallel architecture” Nature Biotechnology22:435-39) or sieve valves. Sieve valves are depicted in FIG. 3. Sievevalves (FIGS. 3A and C) are composed of a square-profile fluidic lineand a regular control membrane, and thus differ from a normal valves(FIGS. 3A and C) based on a round-profile fluidic line. In general, whenvalves operate, the valve membranes deflect in an elliptic shape (FIGS.3 c and d). In the case of normal valve (FIG. 3C), the deflectedmembrane is fully compliant to the round-profile fluidic channel lead tocomplete close of the valve. For a sieve valve (FIG. 3D), a deflectedmembrane partially closes the valve, generating two small gaps the twochannel edges of the square-profile channel. When a solution (e.g., anaqueous solution) containing suspended beads in appropriate sizes isintroduced into the fluidic chambers, the beads are trapped by the sievevalves while the solution is allowed to pass through the closed sievevalve. By using this design, a variety of miniaturized columns filledwith different type of beads (e.g., ion exchange resin, silica gel andC¹⁸ can be achieved for applications such as ion extraction, filtration,purification and chromatography). In one embodiment, the favorabledimensionalities of beads are in the range of 2 μm to as 50 μm dependingon the specific geometry to the channels and valves.

Sieve valves can be constructed using standard multilayer softlithography (MSL) methods (see, e.g., Unger. et al., Science 2000,288:113-16 and patent publications US20040229349; US20040224380; andUS20040072278). For example, a device with sieve valves has beenconstructed of three layers of the silicone elastomerpolydimethylsiloxane (PDMS) (General Electric) bonded to a RCA cleaned#1.5 glass coverslip. The device was fabricated as described in Fu etal., Nat Biotechnol 1999, 17:1109-11 with slight modifications (Studeret al., J. Appl. Phys. 2004, 95:393-98). Negative master molds werefabricated out of photoresist by standard optical lithography andpatterned with 20,000 dpi transparency masks (CAD/Art Services) draftedwith AutoCAD software (Autodesk). The flow layer masks (column portionand channel portion) were sized to 101.5% of the control layer masks tocompensate for shrinking of features during the first elastomer curingstep. The flow master molds were fabricated out of 40 μm AZ-100XT/13 μmSU8-2015 photoresists (Clariant/Microchem) and the control molds werecast from 24 μm SU8-2025 (Microchem).

In order to implement sieve valves, the flow channel portion wherecolumns are to be constructed has a rectangular profile in crosssection. Therefore, in one embodiment, a multistep lithography processis used for microfluidic devices composed of both sieve valves andconventional valves (Unger et al., 2000, Science 288:113-116). In oneapproach, for example, the column resist is spun onto a silicon waferand processed, followed by processing the resist for the conventionalfluid channels. The fabrication of molds having a rounded flow structureis achieved by thermal re-flow of the patterned photoresist. Negativephoto-resists such as SU8 rely on thermal polymerization of UV-exposedregions, and therefore can not be reflowed. In order to be compatiblewith membrane valves, flow channel sections are defined using a positivephotoresist such as AZ-50 (Clariant Corp. Charlotte, N.C.).

Once the fluid channels are processed, the two layer mold is heated(e.g., baked on a hot plate of 200 degrees C. for 2 hours) so that thephotoresist can reflow and form a rounded shape, which is important forcomplete valve closure (see Unger, supra). A hard bake step is alsoimplemented between resist steps, in order to make the column resistmechanically robust for downstream processing. Most devices that havesieve valves also have conventional valves, and have both rounded andnon-rounded (e.g., rectangular) flow channels.

As noted above, the separation device can fluidically communicate withthe reactor. In one embodiment a reactant is concentrated or purifiedand then transported to a reactor. In another embodiment a product isconcentrated or purified and then transported to a reactor. It will berecognized that in a series of chemical reactions some compounds will beboth reactants and products.

Exemplary Microscale (Off-Chip) Columns

In certain embodiments, the microfluidic system includes an off-chipchromatography device such as a microscale column. As used herein and inthis context, off-chip means the column is not integral to the CRC, andspecifically that the column material is not situated within amicrofluidic channel in the device. Thus, a column that is “off-chip” inthis sense can be attached to the chip, placed in a carrier module inwhich the chip is also placed, or fluidically tied to the chip bytubing. In these cases the chromatography column can be removed from thechip without destroying the device.

Advantages of off-chip columns for certain embodiments can includeincreased capacity and increased through-put, due in part to use of acolumn having a larger size (e.g., a microscale column) than can beconveniently fabricated within a microfluidic channel. Microscalecolumns useful in the present devices and methods include (but are notlimited to) columns with a column volume between about 1 microliters andabout 20 microliters, usually between about 5 microliters and about 10microliters.

In certain applications an off-chip design may have advantages. Usingthe synthesis of [¹⁸F]FDG as described in Example 3 as an example,advantages of using a microscale off-chip column include (a) thechannels that supply the target water into the ion exchange column canbe be wider, resulting in much faster loading rates; and (b) the columncapacity can be increased, since at least some resin can be packagedmore tightly than by collecting the beads by filtration. Otheradvantages can include the ability to use a modular cartridge design inwhich a pre-packed ion exchange cartridge is placed on the carriermodule. The off-chip design also allows use and testing of a greatervariety of resins, including resins having a bead size larger than 15microns. In one embodiment the column volume is 2.2 microliters, theexchange resin is AG-1 X8 (200-400 mesh), the dead volume left for thesolvent is <1 uL. Using a prototype column of this design up to 800 mCiof ¹⁸F⁻ could be loaded with 99.5% trapping efficiency from 1.8 mL oftarget water. A release efficiency of 92.7% with 20 uL of 0.05M K₂CO₃was observed. Off-chip columns are also useful when the stationary phaseis destroyed during chromatography (for example, as inacid-neutralization chromatography using an alumina column). Columns canbe replaced and devices reused.

It will be appreciated that a particular microfluidic system may haveone or more (up to several hundreds or more) on-chip columns, may haveone or more off-chip columns, and may employ both on-chip and off-chipcolumns. It will also be clear that CRCs may include multiple columns,which may have different functions in a chemical reaction or otherprocess, and may contain different resins or other chromatographymaterial.

Pumps

Microfluidic devices of the present invention may include one or moreintegral pumps for transport of fluids through flow channels and intoand out of other device components (e.g., column or reactors) or thedevice itself. Suitable pumps can be electronic, electrostatic,magnetic, mechanical, syringe, pneumatic, or peristaltic. Preferablyperistaltic pumps, such as those described in U.S. Pat. No. 6,408,878B2, are used. Alternatively pumps can be external to the chip. Pumps arealso used to transport fluids (e.g., water) through control channels toactuate valves or guard channels to minimize evaporation is selectedregions of the chip. Pumps are also used to draw a vacuum in, forexample, vent channels.

Temperature Control Components

In certain embodiments, a solvent, reaction mixture, reagent or productis heated or cooled to initiate, maintain or optimize a reaction orpreserve the reagent or product. Thus, the devices and systems of theinvention may include temperature control systems that modulatetemperature of the entire device or a particular region or component ofthe device (e.g., reservoir or reactor). Examples of suitabletemperature control systems include, but are not limited to, Peltierdevices, resistive heaters, heat exchangers and an indium tin oxideelement (see e.g., U.S. Pat. No. 6,960,437 B2). Solutions also can beheated using a light source such as a laser. A number of differentoptions of varying sophistication are available for controllingtemperature within selected regions of the microfluidic device or theentire device. Thus, as used herein, the term temperature controller ismeant broadly to refer to a device or element that can regulatetemperature of the entire microfluidic device or within a portion of themicrofluidic device.

In some embodiments, it is useful to incorporate sensors detectingtemperature at various regions of the device. One structure fordetecting temperature is a thermocouple. Such a thermocouple could becreated as thin film wires patterned on the underlying substratematerial, or as wires incorporated directly into the microfabricatedelastomer material itself. Temperature can also be sensed through achange in electrical resistance. For example, change in resistance of athermistor fabricated on an underlying semiconductor substrate utilizingconventional techniques can be calibrated to a given temperature change.Alternatively, a thermistor could be inserted directly into themicrofabricated elastomer material. Still another approach to detectionof temperature by resistance is described in Wu et al., 2001, “MEMS FlowSensors for Nano-fluidic Applications”, Sensors and Actuators A 89152-158. Thermo-chromatic materials

Monitoring Components

In certain embodiments, the microfluidic system or device includesmonitoring devices and signal detectors. Exemplary signal detectorsmonitor visible, fluorescent, and UV light (intensity, scattering,absorption) luminescence, differential reflectivity, electricalresistance, resistivity, impedance, and voltage. Applications can alsoutilize scintillation proximity assay techniques, confocal laserscanning, radiochemical detection, fluorescence polarization and othermethods.

Control Channels

A “control channel” is a channel separated from a flow channel by anelastomeric membrane that can be deflected into or retracted from theflow channel in response to an actuation force (i.e., acting as a“valve”). The dimensions of control channels can vary widely buttypically include at least one cross-sectional dimension (e.g., height,width, or diameter) less than 1 mm, preferably less than 0.5 mm, andoften less than 0.3 mm. For example, in one embodiment, a controlchannel has dimensions of 250 micrometers wide by 250 micrometers high.In another embodiment, a control channel has dimensions of 300micrometers wide by 50 micrometers high. See, Unger et al., 2000,Science 288:113-116; US 2004/0115838; and PCT publications WO 01/01025;WO 2005030822 and WO 2005084191.

Guard Channels

In certain embodiments, the microfluidic device contains guard channelsin an elastomeric layer. Guard channels are channels formed within aelastomeric device through which a solution (e.g., water) can be flowedto increase the water vapor pressure within the elastomeric material,thereby reducing evaporation at selected portions of the device and/orat selected times in a reaction series. Guard channels are described inU.S. Patent Application Publication No. 2003/0138829.

Vias

A “via” refers to a channel formed in an elastomeric device to providefluid access between an external port of the device and one or more flowchannels. Thus, a via can serve as a sample input or output, forexample. Also encompassed are “vertical vias” or interconnects betweenthe elastomer layers (which may be created by lithographicallypatterning an etch resistant layer on top of a elastomer layer, thenetching the elastomer and finally removing the etch resist before addingthe last layer of elastomer). See U.S. Pat. No. 6,408,878 and patentpublication US20050166980.

Other properties of individual reactors will be apparent on review ofthis disclosure. For example, in most embodiments of the invention thereactor is free of cells, cellular material, or nucleic acids, and/oroccurring polypeptides from a biological source (cell, virion, etc.).

Section 4. Synthesis of a Reaction Product

As discussed above, the invention provides methods for carrying out achemical reaction, or a series of chemical reactions, using amicrofluidic device. In a basic aspect, the method involves introducinga solution containing one or more reactants into a microfluidic reactor,then fluidically isolating the reactor, and then removing solvent fromthe fluidically isolated reactor. A number of optional additional stepsare described below. In particular, methods for effecting solventexchanges, conducting sequential chemical reactions in the reactor, andfunctionally integrating various components of the microfluidic deviceare described.

In a first basic aspect, a solution containing a reactant in a solventsystem is introduced into the reactor via microfluidic flow channels.For ease of reference, this is a first solution containing a firstreactant in a first solvent system. The reactor is fluidically isolatedand all or a portion of the solvent system is removed from thefluidically isolated chamber while retaining the first reactant. Thereactor is fluidically joined to a channel, and a second solvent systemis introduced in which the reactant is resolubilized. The second solventsystem may contain a reactant or catalyst, as described below.Alternatively, the reactant in solution in the second solvent system canbe removed from the reactor and transported to a different reactor,separation column, or the like, to undergo further chemical processes.

In another basic aspect, two different solutions, each containing areactant in the chemical reaction, are introduced into the reactor viamicrofluidic flow channels. For ease of reference, the first solutioncontains the first reactant in the first solvent system, and the secondsolution contains the second reactant in the second solvent system. Thefirst and second solvent systems may be the same or different, and eachmay consist of a single solvent (e.g., acetonitrile) or a combination ofsolvents (e.g., acetonitrile and water). Each solution optionallycontains additional solutes, including, in some cases, additionalreactants.

Introducing a solution into a reactor can be accomplished using anyfluid transport mechanism suited to the particular microfluidic device.Typically solutions are pumped (e.g., with a peristaltic pump). The twosolutions can be added simultaneously or in either order using any of anumber of filling strategies. In one strategy, two solutions areintroduced simultaneously, and the reactor is then fluidically isolated.In a second strategy, the first solution is introduced to partially fillthe reactor, the second solution is then added to further (or completelyfill the reactor) and the reactor is then fluidically isolated. Thisstrategy is illustrated in Example 3, below, in which, inter alia, thereactor is two-thirds filled with a mixture of ¹⁸F⁻/Kryptofix222/K₂CO₃(in MeCN), and then mannose triflate (in MeCN) is introduced to fill thereactor. In a third strategy, the reactor is completely filled with thefirst solution, the second solution is then introduced, displacing aportion of the first solution, and the reactor is then fluidicallyisolated. In a fourth strategy, the reactor is completely filled withthe first solution, the reactor is isolated, all or some of the firstsolvent system is withdrawn, the reactor is fluidically joined, thesecond solution is introduced, and the reactor is again fluidicallyisolated. This fourth strategy is illustrated in Example 1, below, inwhich, inter alia, (1) an aqueous solution containing ¹⁹F—KF wasintroduced into the reactor (reaction loop), (2) the reactor wasfluidically isolated, (3) water was removed from the reactor willretaining the KF, (4) the reactor was fluidically joined, (5) D-mannosetriflate and Kryptofix 222 were added, and (6) and the reactor was againfluidically isolated. Introduction of solutions into the reactor can befacilitated using vent channels to withdraw gas (e.g., air) that mayoccupy the reactor before filling (see Example 3, FIG. 12).

Additional solvents or solutions containing additional reactants,catalysts, buffers, reagents, reaction components, and the like can beintroduced into the reactor via flow channels at any time the reactor isnot fluidically isolated, including before, during and/or after theintroduction of the first solution and/or before, during and/or afterintroduction of the second solution. Addition may be in any orderconsistent with the chemistry of the desired reaction. Further, it willbe appreciated that, as illustrated in the examples, the reactor can befluidically isolated or joined at various times in the filling processto facilitate filling.

Removal of a Solvent System

The invention provides a method for solvent exchange in which a solventsystem is removed from a reactor that is permeable to a solvent invapor, but not liquid, form. In certain embodiments the method includesa step of removing all or a portion of a solvent system (or individualcomponent solvents) from the fluidically isolated reactor whileretaining, and optionally without removing, a solute, such as a reactantor product, from the reactor. Solvent is removed by evaporation througha gas permeable portion of the reactor chamber wall. As discussed inSection 3, above, at least a portion of the reactor wall is permeable tothe vapor form of a solvent, but not the liquid form, allowing vapor toescape a fluidically isolated reactor. As discussed in Section 3 anexemplary reactor is fabricated at least in part from a gas permeableelastomeric material. Exemplary elastomers include polydimethylsiloxaneand perfluoropolyether, which are characterized by outstanding gaspermeability. In one embodiment the reactor is fabricated entirely orsubstantially from elastomer. In some embodiments, the rate ofevaporation is accelerated by application of heat and/or use of ventchannels.

The presence of gas permeable materials in the reactor allows the userto (i) carry out solvent exchange(s) in the reactor; (ii) concentratereactants, products and other solutes in the reactor; (iii) change thecomposition of solutions in the chamber (e.g., by differentiallyevaporating different solvents in a solvent system, based on differentboiling points); and carry out other useful processes.

Solvent exchange can be accomplished by removing a first solvent from afluidically isolated reactor, fluidically joining the reactor to flowchannel(s) by opening a valve or valves, and introducing a secondsolvent into the reactor via a flow channel. The valve(s) can then beclosed to again isolate the reactor. Additional reaction components(such as reactants) can also introduced and may be, for example,introduced in solution with the solvent system. Example 1 illustratessolvent exchange. As described, aqueous ¹⁸F-fluorine was introduced intothe reactor, the reactor isolated, the water (solvent) was removed byheating, and a different solvent, acetonitrile, was then introduced intothe reactor. It is not necessary, and is sometimes undesirable, toremove all of a first solvent during a solvent exchange step (forexample, if reactants are very difficult to resoluablize). A solventexchange step can involve removal of at least about 5%, at least about10%, at least about 25%, at least about 50%, at least about 75%, atleast about 80%, at least about 90%, at least about 95%, at least about99%, or all of a solvent contained in a reactor.

In one aspect the invention provides a method for removing solvent froma reaction chamber (reactor) of a microfluidic device by (i) providing amicrofluidic device comprising a reactor that contains a solute compoundand a solvent system and (2) removing at least 25%, preferably at least50% of the solvent system from the reactor while retaining the solutecompound in the reactor, whereby the amount of the solute compound inthe reactor per unit volume of the solvent system in the reactor isincreased. In one embodiment the solute compound remains in solution andthe concentration of the solute in the solution is increased. In oneembodiment the solvent is other than water. In one embodiment thesolvent is water.

In one embodiment of this method the solute compound is a radionuclideor a molecule comprising a radionuclide. The radionuclide can be, forexample, [¹¹C], [¹²⁴I], [¹⁸F], [¹²⁴], [¹³N], [⁵²Fe], [⁵⁵Co], [⁷⁵Br],[⁷⁶Br], [⁹⁴Tc], [¹¹¹In], [⁹⁹Tc], [¹¹¹In], [⁶⁷Ga], [¹²³I], [¹²⁵I], [¹⁴C],or [³²P]. Preferably the solute compound is [¹⁸F]fluoride or a moleculecomprising [¹⁸F]fluoride such as [¹⁸F]-potassium fluoride. In someembodiments the solute compound is a cryptand, such as Kryptofix 222. Inembodiments, the solute compound is i)2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose; ii)2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrile;iii) D-mannose triflate; a compound listed in Table 1. In preferredembodiments of the invention, more than one solute compound is retainedin the reactor. For example, 2, 3, 4, 5, 6 or more than 6 solutes may beretained in the reactor.

Generation of the Reaction Product

In certain embodiments, the reactor is maintained in a fluidicallyisolated state (e.g., valves are kept closed) for a time and underconditions sufficient for a reaction product to accumulate in thereactor.

For certain combinations of reactants and reaction conditions, little orno product will be formed until sufficient time has passed for thereaction to take place (as determined by reaction kinetics) and/or thereaction is initiated or accelerated by a change in the environment ofthe fluidically isolated reactor (e.g., heating of the reactionmixture).

In other cases, product may begin to form even before the reactor isisolated, such as soon as the reactants are intermixed, or uponintroduction of a component that changes the reaction environment (achange in the solvent system, introduction of reactants, catalysts,salts, buffers, ions, etc.). If the reaction is rapid, product may becompletely generated before the reactor is isolated. In one embodiment,a substantial amount of the reaction product is produced prior to thestep of fluidically isolating the reactor. By “substantial amount” ismeant more than 80%, preferably more than 90%, and sometimes more then95% of the total yield of the reaction. In another embodiment, aninsubstantial amount of the reaction product is produced prior to thestep of fluidically isolating the reactor. By “insubstantial amount” ismeant less than 20%, preferably less than 10%, and sometimes less than5% of the total yield of the reaction. If the reaction is less rapid, aportion of the product may be generated before the reactor is isolatedand product will continue to accumulate for a period after the reactoris isolated). In any event, the reactor is maintained in a fluidicallyisolated state for a time and under conditions sufficient for a reactionproduct to accumulate in the reactor. If, as a limiting example, thereactants react immediately and completely when introduced into thereactor and prior to fluidic isolation of the reactor, “maintaining thereactor in a fluidically isolated state for a time and under conditionssufficient for a reaction product” includes a “zero time;” that is,accumulation of the reaction product is complete at the time the reactoris isolated.

As noted above, for many reactions little or no product will be formeduntil the reaction is initiated or accelerated by a change in theenvironment such as heating of the reaction mixture. Advantageously, asolution inside a microfluidic reactor fabricated, entirely or in part,from certain elastomers (e.g., PDMS), can be heated above normal(atmospheric) boiling point or the solvent or solution, generating ahigh-temperature and high-pressure reaction environment. As a result,the reaction kinetics are accelerated, and reaction time can beshortened. Pressure is mediated not only by the heat supplied to thechip, but also by the porosity of the elastomer matrix. In this example,PDMS plays a role akin to the safety valve of a pressure cooker thatregulates the “cooking pressure” within a critical range.

Mixing Solvents, Reactants and Reagents

Chemical reactions and other chemical processes may require that thereaction components be actively mixed, or may be accelerated with activemixing. Several methods for mixing are described below, for illustrationand not limitation. Method (a) is particularly relevant to a reactorconfigured as a loop (including non-circular structures described inU.S. Pat. No. 6,767,706). Methods (b)-(g) are particularly relevant toreactors that have a large volume, such as a coin-shaped reactor.

a) Loop Channel Mixing

Solutions can be mixed in a loop channel (or “circulation loop channel”)by introducing the solutions into the loop channel and activating a pump(e.g., peristaltic pump) associated with the loop channel to circulateat least one of the different fluids about the circulation loop channelto mix the different fluids. See U.S. Pat. No. 6,767,706.

b) Bottom-Up Mixing

Mixing can be achieved by causing the membrane of the large flat rounddead end channel (denoted “control channel” in the figure) below thereactor to be actuated (i.e., expand and contract) at a certainfrequency as illustrated in FIG. 19. This method has certaindisadvantages: (i) There is little room left in the control channellayer for the evaporation radiator (if a bottom vent location is used);(ii) The membrane between the control channel and the reaction chambermay collapse during the final curing of the chip because of largesurface area/thickness ratio; (iii) The mixing efficiency may belimited; (iv) Complete elution of product from the reactor may bedifficult; (v) The problem of leaving some product behind in the reactorafter final elution still exists; (vi) The dead-end channel takes upvaluable space in the (in some embodiments) control channel layer.

c) Pulse Mixing

In one approach to mixing, a dead-end serpentine channel is positionedunderneath the reactor, e.g., in the control layer underneath thereaction chamber. The dead-end channel can be filled with liquid (e.g.,Krytox® oil, Dupont) and serve as a mixer by introducing waves in thereactor by pulsing the pressure applied to this channel. This system canalso be used to remove product from the reactor (e.g., the same wavesare oriented towards the exit channel of the reactor). To empty thereactor the serpentine channel is actuated to generate wave. Inaddition, gas (e.g., N₂) can be introduced into a vent channel (insteadof vacuum) to building up pressure inside the reactor. FIG. 20illustrates a double radiator system in which the vent channel (shownwith N₂) and the liquid-filled dead-end channel (“Krytox”). This figurealso illustrates another exemplary vent channel configuration and thearrangement of multiple systems adjacent to a reactor and in the sameelastomeric layer (a “double radiator” configuration).

d) Chemically Promoted Mixing (Self-Stirring Reaction)

Mixing is chemically promoted when a reaction causes turbulence orotherwise results in movement of solutions. For example, in the [¹⁸F]FDGsynthesis described elsewhere herein, mixing of [¹⁸F]FTAG solution withHCl in Reaction step II is “chemically promoted” by having the twosolutions engage in a vigorous acid-base reaction at the interface. Thisproduces swirling, which in turn rapidly mixes the two solutions. Thistype of mixing mechanism involves no additional features in the device,but relies on the choices of reactants and reaction steps. Note that inthe [¹⁸F]FDG synthesis, mixing of two solutions is only needed in thehydrolysis step, because in all other steps when a reagent solution isintroduced into the reaction chamber, all other reagents are alreadydistributed throughout the chamber in the solid form. As notedelsewhere, if the FTAG solution is evaporated to dryness, it isdifficult to resolubilize.

e) Vacuum-Compression Mixing

In a coin shape reactor (for example) made from elastomeric material,the walls of the reactor chamber can collapse inward under certaincircumstances. For example, in the [¹⁸F]FDG synthesis, as theacetonitrile is evaporated from the FTAG solution in a closed reactionchamber, the coin-shape of the latter allows its flat surfaces to cavein as the volume of the solution is reduced (and a vacuum is createdinside the chamber). When the valve on the acid channel is opened, theelastomer restores its shape and volume of the chamber by pulling theacid inside rapidly. The speed of such introduction of the secondsolution promotes virtually instantaneous mixing.

f) Expansion Mixing

Another mixing mechanism, suitable for elastomeric and similar devicestakes advantage of the elasticity of the material from which the chip isfabricated. Such mixing starts by having the reactor half-full with asolution of one reagent. Subsequent introduction the second reagentfills the empty half of the chamber. In order to “stir” the reaction,the pressure can be pulsed in the flow channel used for the introductionof the second reagent (while the valve on that channel is open). Theelastic chamber will expand and return to its shape at the frequency ofpulsing. The contents of the chamber will then move out of the chamberand back in rapidly resulting in rapid and complete mixing followed byclosing the corresponding valve on the channel used.

g) Mixing by Introduction

The spatial relationship between flow channels (such as distributionmanifolds) and reactors can be selected to accelerate mixing or fluidmovement within an isolated microfluidic environment. For example, theuse of a distribution manifold for simultaneous introduction of asolution into the chamber can result in efficient mixing.

i) Other Mixing Methods

The mixing methods above are provided as examples, and not forlimitation. A variety of other methods for mixing will be apparent tothe practitioner upon review of this disclosure.

Sequential Reactions

The device of the invention is particularly suited for carrying outsequential reactions (in which a product of a first reaction is areactant in a subsequent reaction). Thus, for example, the inventionprovides a method for carrying out sequential chemical reactionsproviding a microfluidic device comprising a reactor, providing reagentssufficient for carrying out at least two sequential chemical reactions,carrying out a first chemical reaction in the reactor, thereby producinga first reaction product; and carrying out a subsequent chemicalreaction in the reactor, where the first reaction product is a reactantin the second chemical reaction and where the first reaction product isnot removed from the reactor prior to the second reaction. In a relatedembodiment the first reaction product is a catalyst in a subsequentreaction. In one embodiment of the invention the two reactions arecarried out in different solvent systems. In one embodiment, at least aportion of the solvent system in which the first reaction occurs isremoved from the reactor while retaining, optionally without removing,the first product, by evaporating the solvent system out of thefluidically isolated reactor.

In a closely related embodiment the method of carrying out sequentialchemical reactions include reacting a first reactant and a secondreactant in the reactor, where the first and second reactants are insolution in a reaction solvent system, where the reactor is fluidicallyisolated, and where a first reaction product is produced; evaporating atleast a portion of the reaction solvent system from the fluidicallyisolated reactor; introducing into the reactor a solution containing athird reactant, while retaining, optionally without removing, the firstproduct in the reactor. In an embodiment, the first reaction product andthe third reactant react to product a new (second) reaction product. Inanother related embodiment the method of carrying out sequentialchemical reactions include reacting a first reactant and a secondreactant in the reactor, where the first and second reactants are insolution in a reaction solvent system, where the reactor is fluidicallyisolated, and where a first reaction product is produced; evaporating atleast a portion of the reaction solvent system from the fluidicallyisolated reactor; introducing into the reactor a solution containing acatalyst, while retaining, optionally without removing, the firstproduct in the reactor. In an embodiment, the first reaction productreacts with a third reactant, which is either present in the reactor oradded, in a reaction catalyzed by the catalyst, to product a new(second) reaction product. In another embodiment, the first reactionproduct does not combine with another reactant, but is instead modified(e.g., hydrolyzed, deprotected, etc.) in the presence of the catalyst. Areaction series of this type (Reactions I and II) resulting inproduction of [¹⁸F]FDG is described in the Examples, below.

¹⁸F-fluoride+mannosetriflate→2-deoxy-2-18F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose  ReactionI

2-deoxy-2-18F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose+HCl→¹⁸F-2DG  Reaction II

As described, Reaction I was carried out in a reactor in acetonitrile(the reaction solvent system). At the completion of Reaction I, theacetonitrile was removed from the fluidically isolated reactor byevaporation through the PDMS wall. An aqueous solution containing HClwas introduced into the chamber and the [¹⁸F]FDG product generated inReaction II. In this reaction HCl acts as a catalyst. Catalysts that maybe used in reactions include heterogeneous catalysts (present indifferent phases from the reactants, e.g. a solid catalyst in a liquidreaction mixture) and homogenous catalysts (present in the same phase,e.g. dissolved catalyst in a liquid reaction mixture). In an embodimentthe catalyst is an acid.

It will be appreciated that, if desired, additional reactions can becarried out in sequence by repeating rounds of reaction, solventmodification or removal, and addition of reagents such as reactants,catalysts or reaction initiators. In one embodiment, three sequentialreactions are carried out, such as:

R1+R2→P1  1.

P1+R3→P2  2.

P2+R4→P3  3.

where R1-4 are reactants and P1-3 are reaction products (P1-2 are alsoreactants). Solvent exchange or modification can take place, forexample, between Steps 1 and 2 and between Steps 2 and 3. In someembodiments, parallel reactions (i.e., generation of more than oneproduct in the reactor) take place, e.g.,

R1+R2→P1;R3+R4→P2  1.

P1+P2+catalyst→P3  2.

with a solvent exchange or modification between Steps 1 and 2. In otherembodiments multicomponent or combinatorial reactions are carried out.For example, and not limitation, exemplary reactions that can be carriedout in the device of the invention are described below.

Although it is convenient to carry out sequential reactions in the samechamber, sequential reactions using the methods of the invention canalso be carried out by conducting a first reaction in a first reactor ina first solvent, transporting the product of the reaction to a secondreactor and conducting a second reaction in the second reactor in asecond solvent, with solvent exchange occurring in either the first orsecond reactors.

In another scheme, two or more sequential reactions can be carried outin a first reactor in which solvent exchange occurs and the product thentransported out of the reactor and modified, and the “modified product”transported to a second reactor. Modifications include, for example,chemical modification, concentration (e.g., by column chromatography),mixing with other agents (e.g., in a rotary mixer), heating, and variousother modifications that will be apparent to the ordinarily skilledchemist.

A wide variety of chemical processes may be integrated into themicrofluidic devices of the present invention and adapted for sequentialsynthetic processes on the nanogram scale. Examples include, forillustration, oxidation, reduction, esterification, hydrolysis,substitution, Suzuki couplings, Kumada couplings, nitrations, diazocouplings, diazotizations, photocyanations, dehydration reactions,esterifications, fluorinations, hydrolysis reactions, Grubbs metathesis,Kumada-Corriu coupling, aldol reactions, and oxidations. See CurrentOpinion in Chemical Biology 7:380-387 (2003), and de Mello et al., 2002Lab Chip, 2:7N-13N. In one embodiment the chemical reaction is asubstitution reaction. In one embodiment the substitution is afluorination reaction.

Additional specific examples include of peptide syntheses, the synthesisof a series of 2-aminothiazoles using a Hantzsch synthesis, thesynthesis of cycloadducts in a condensation of an aldehyde with an EDDAcatalyst, Swern oxidation, and labeling of carboxylic esters with ashort-lived positron-emitter (e.g., carbon-11 or fluorine-18), andoxidative dehydrogenation of alcohols. See Current Opinion in ChemicalBiology 7:380-387 (2003), Kawaguchi et al., 2005, Angew. Chem. Int. Ed.,44:2413-16, and Lu et al., 2004, Lab Chip 4: 523-25; de Mello et al.,2002, Lab Chip, 2:7N-13N.

In one embodiment, the reaction is carried out in the presence of acryptand. For illustration and not limitation exemplary cryptandsinclude Kryptofix 5; Kryptofix 21; Kryptofix 22; Kryptofix 22 azatrisulfate; Kryptofix 22 DD; Kryptofix 22 polymer; Kryptofix 23;Kryptofix 111; Kryptofix 211; Kryptofix 221; Kryptofix 221 B polymer;Kryptofix 222; Kryptofix 222 B; Kryptofix 222 BB; Kryptofix 222 Bpolymer; Kryptofix 222 CC; Kryptofix 222 D; Crown ether/12-Crown-4;Crown ether/15-Crown-5; 811684 Crown ether/18-Crown-6; Crownether/4′-Nitrobenzo-15-crown-5; Crown ether/Decyl-18-crown-6; Crownether/Dicyclohexyl-18-crown-6; Crown ether/N-Phenylaza-15-crown-5. Allof these are commercially available (Merck KGaA). In one embodiment thecryptand is Kryptofix 222[4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane].

These reactions may be adapted to sequential syntheses performed in themicrofluidic device of the present invention. The compounds generated bysuch reactions can be composed of any components that can be joined toone another through chemical bonds in a series of steps. Thus, thecomponents can be any class of monomer useful in combinatorialsynthesis. Hence, the components, monomers, or building blocks caninclude, but are not limited to, amino acids, carbohydrates, lipids,phospholipids, carbamates, sulfones, sulfoxides, esters, nucleosides,heterocyclic molecules, amines, carboxylic acids, aldehydes, ketones,isocyanates, isothiocyanates, thiols, alkyl halides, phenolic molecules,boronic acids, stannanes, alkyl or aryl lithium molecules, Grignardreagents, alkenes, alkynes, dienes and urea derivatives.

Given the diversity of components that can be utilized in the methods ofthe invention, the compounds capable of being formed are equallydiverse. Essentially molecules of any type that can be formed inmultiple cycles in which the ultimate compound or product, is formed ina component by component fashion can be synthesized according to themethods of the invention. Examples of compounds that can be synthesizedinclude but are not limited to molecular imaging agents,benzodiazepines, thiazolidinones, and imidizolidinones. The finalcompounds can be linear, branched, cyclic or assume other conformations.The compounds can be designed to have potential biological activity ornon-biological activity.

In one aspect, the invention provides a method for radiolabeling acompound. Sequential radiolabeling processes that can be carried out ona automated integrated microfluidic CRC include, for example, one ormore of the following: (i) concentration or pre-treatment of radioactivereagent or precursor, (ii) mixing and reacting of radiolabeling reagentwith the precursor to produce radiolabeled intermediate, (iii)deprotection or chemical modification of the radiolabeled intermediate,(iv) purification of radiolabeled product which is the desired molecularprobes and (v) quality analysis and control.

In one aspect, the invention provides a method for synthesizing aradiolabeled product in a microfluidic environment by mixing andreacting of a radiolabled reactant with a precursor reactant compound toproduce a radiolabeled product, where the mixing and reacting occurs ina microfluidic reactor and where the radiolabled reagent is introducedinto the reactor in a first solvent and the radiolabeled precursor isintroduced in a second solvent that is different from the first. In someembodiments, the radiolabeled product is a radiolabeled molecularimaging probe. In some embodiments the radiolabeled molecular imagingprobe is 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG);6-[¹⁸F]fluoro-L-3,4-dihydroxyphenylalanine ([¹⁸F]FDOPA);6-[¹⁸F]fluoro-L-meta-tyrosine ([¹⁸F]FMT),9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butyl]guanine ([¹⁸F]FHBG),9-[(3-[¹⁸F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([¹⁸F]FHPG),3-(2′-[¹⁸F]fluoroethyl)spiperone([¹⁸F]FESP),3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT),4-[¹⁸F]fluoro-N-[2-[1-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyr-idinyl-benzamide([¹⁸F]p-MPPF),2-(1-{6-[(2-[¹⁸F]fluoroethyl)(met-hyl)amino]-2-naphthyl}ethylidine)malononitrile([¹⁸F]FDDNP), 2-[¹⁸F]fluoro-alpha-methyltyrosine,[¹⁸F]fluoromisonidazole([¹⁸F]FMISO),5-[¹⁸F]fluoro-2′-deoxyuridine([¹⁸F]FdUrd), [¹¹C]raclopride,[¹¹C]N-methylspiperone, [¹¹C]cocaine, [¹¹C]nomifensine, [¹¹C]deprenyl,[¹¹C]clozapine, [¹¹C]methionine, [11C]choline, [¹¹C]thymidine,[¹¹C]flumazenil, [¹¹C]alpha-aminoisobutyric acid or a protected form ofone of the foregoing compounds. In particular embodiments theradiolabeled reactant is [¹⁸F]-potassium fluoride and the precursorreactant is2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrileor is D-mannose triflate. Table 1 shows, for example and not forlimitation, exemplary PET imaging agents and exemplary precursors. Itwill be appreciated that multiple synthetic routes can be used forsynthesis of such agents.

TABLE 1 precursor(s) product D-mannose triflate. [¹⁸F]FDG2-(1-{6-[(2-[(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2- [¹⁸F]FDDNPnaphthyl}ethylidine)malononitrile,N-Boc-5′-O-dimethoxytrityl-3′-O-(4-nitrophenylsulfonyl)- [¹⁸F]FLTthymidine N²-(p-anisyldiphenylmethyl)-9-[(4-p-toluenes-ulfonyloxy)-3-(p-[¹⁸F]FHBG anisyldiphenylmethoxymethyl)butyl]guanineN²-(p-anisyldiphenylmethyl)-9-[[1-[(.beta.-anisy- [¹⁸F]FHPGldiphenylmethoxy)-3-(p-toluenesulfonyloxy)-2- propoxy]methyl]guanine8-[4-(4-fluorophenyl)-4,4-(ethylenedioxy)bu-tyl]-3-[2′-(2,4,6- [¹⁸F]FESPtrimethylphenylsulfonyloxyethyl)]-1-phenyl-1,3,8-triazas-piro[4.5]decan-4-one 5′-O-Boc-2,3′anhydrothymidine [¹⁸F]FLTN-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethy-l]-4-nitro-N-2- p-[¹⁸F]MPPFpyridinyl-benzamide 1,2-bis(tosyloxy)ethane and N,N-dimethylethanolamine[¹⁸F]fluoroethylcholine; Ditosylmethane and N,N-dimethylethanolamine[¹⁸F]fluorocholine

Methods for synthesis of these compounds are well known and can beeasily adapted to use in the microfluidic systems of the invention (see,e.g., Hamacher et al., 1986, “Efficient Stereospecific Synthesis ofNo-Carrier-Added 2-[F-18]-Fluoro-2-Deoxy-D-Glucose Using AminopolyetherSupported Nucleophilic-Substitution” Journal of Nuclear Medicine27:235-238; Padgett et al., 1989, “Computer-Controlled RadiochemicalSynthesis—a Chemistry Process-Control Unit for the Automated Productionof Radiochemicals” Applied Radiation and Isotopes 40:433; Machulla etal., 2000, “Simplified labeling approach for synthesizing3′-deoxy-3′-[F-18]fluorothymidine ([F-18]FLT)” Journal of Radionalyticaland Nuclear Chemistry 243:843-46; Shoghi-Jadid. et al., 2002,“Localization of neurofibrillary tangles and beta-amyloid plaques in thebrains of living patients with Alzheimer disease” American Journal ofGeriatric Psychiatry 10:24-35).

The skilled artisan will immediately appreciate that the invention couldbe readily adapted for synthesis of any appropriate radioactive compoundcomprising a radionuclide, including radiochemicals useful in otherimaging systems. Radionuclides that may be used include, but are notlimited to, positron emitting radionuclides such as ¹¹C(t,_(1/2)=20.1min), ¹⁸(t,_(1/2)=110 min), ¹²⁴I (t^(1/2)=4.2 days), ¹³N (t_(1/2)=19.3min) and ¹⁵O (t_(1/2)=2.03 min) and other suitable radionuclides, e.g.,⁵²Fe (t_(1/2)=8.3 hr), ⁵⁵(t_(1/2)=17.5 hr), ⁵⁵Co (t_(1/2)=9.7 min),⁷⁵Br(t_(1/2)=98 min), ⁷⁶Br (t^(1/2)=16.1 hr) and ⁹⁴Tc ¹¹¹In=53 min) canbe introduced into molecules to serve as molecular probes for PET.Similarly, the integrated microfluidic CRCs can be applied forintroducing gamma emitters such as ⁹⁹Tc, ¹¹¹In, ⁶⁷Ga, ¹²³I and ¹²⁵I andbeta emitters such as ¹⁴C and ³²P into the respective molecular probes.

For example, in some embodiments, the present invention is directed tohighly efficient incorporations of [¹¹C]-containing molecules, forexample, [¹¹C]methane, [¹¹C]carbon dioxide, [¹¹C]carbon monoxide,[¹¹C]-containing halides ([¹¹C]-RX), [¹¹C]-containing acid chloride(R[¹¹C]COX), (¹¹C]-containing carboxylic acids (R[¹¹C]COOH),[¹¹C]-containing; ester (R[¹¹C]COOR), [¹¹C]-containing alcohols(R[¹¹C]COH), [¹¹C]CN, [¹¹C]CCI4, [¹¹C]phosgene and, [¹¹C]urea into,e.g., PET molecular probes. In some embodiments, the present inventionis directed to highly efficient synthesis of [¹²⁴I]-labeled PETmolecular probes.

Generally, a radioactive isotope is reacted in an appropriate solvent,such as polar aprotic solvents, such as acetonitrile, acetone,1,4-dioxane, tetrahydrofuran (THF), tetramethylenesulfone (sulfolane),N-methylpyrrolidinone (NMP), dimethoxyethane (DME), dimethylacetamide(DMA), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), andhexamethylphosphoramide (HMPA). For solutions containing ¹⁸F, theradioactive isotope is typically in the form of a coordination compoundincluding a phase transfer catalyst and salt complex. One common ¹⁸Fsolution includes Kryptofix 2.2.2 as the phase transfer catalyst and ¹⁸Fin a salt complex with potassium carbonate (K₂CO₃).

In some embodiments, the radiochemical synthesis reaction used in theinvention comprises the additional step of deprotecting theradiochemical following reaction with the radioactive isotope.Typically, the deprotecting step is a hydrolysis reaction that includescontacting and reacting the radiochemical with a hydrolyzing agent, suchas an aqueous base solution or an aqueous acid solution. The aqueousbase solution may be an alkali metal hydroxide (e.g., sodium hydroxideor potassium hydroxide) and the aqueous acid solution may includehydrochloric acid.

In some embodiments, for radionuclide labeling processes, a numbersequential steps may be performed within the microfluidic device. Forclarity, synthesis steps are set forth below using the [¹⁸F]fluorideion, but are equally applicable to other radionuclides usingmodifications apparent to those skilled in the art. It will beappreciated that not all of these steps are required for each synthesis(e.g., intermediate and post-synthetic purification steps may beomitted) and not all of the steps recited are carried out on themicrofluidic system or device of the invention. These steps may include:receiving aqueous [¹⁸F]fluoride ion from the cyclotron target;separating the [¹⁸F]fluoride ion from the water and collecting thewater; generating a solution of reactive [¹⁸F]fluoride ion in an organicand/or polar aprotic solvent (acetonitrile, DMF, DMSO, etc.); providinga solution of a reactive precursor in an organic and/or polar aproticsolvent (acetonitrile, DMF, DMSO, etc.); reacting the [¹⁸F]fluoride ionwith the precursor using a nucleophilic substitution reaction to createa new carbon-fluorine bond, using heat if necessary; purifying theinitial [¹⁸F]fluorinated product by solid phase extraction orchromatography; reacting the purified initial [¹⁸F]fluorinated productwith a second reagent to generate the final [¹⁸F]fluorinated product(e.g., hydrolysis of protecting group(s), if necessary); purifying thefinal [¹⁸F]fluorinated product by, for example, solid phase extractionor chromatography; desolvating the [¹⁸F]fluorinated product; assayingthe purified final [¹⁸F]fluorinated product for radioactivity, UVabsorbance, and conductivity/pH; delivering the purified final[¹⁸F]fluorinated product; and/or dispensing the purified final[18F]fluorinated product

Multicomponent Reactions

In addition to the introduction into the reactor of two solutions eachcontaining a single reactant, it is contemplated that a number of othertypes of reactions will be carried out.

In one embodiment, the first and second reactants are introduced intothe reactor as single solution containing both reactants in a firstsolvent system, and a catalyst of the reaction is added in secondsolvent system. After the introduction of the reactants and catalyst,the reactor is fluidically isolated.

In another embodiment, the first and second reactants are introducedinto the reactor as single solution containing both reactants in a firstsolvent system, and a compound that is neither a catalyst nor areactant, but which affects the environment of the reactor (e.g., causesa change in pH) in a way that initiates or accelerates the reactionbetween the first and second reactants is added. After the introductionof the reactants and compound, the reactor is fluidically isolated.

In a third embodiment, the product is generated in a multicomponentreaction and multiple reactants are introduced into the reactor.Multicomponent reactions are convergent reactions, in which three ormore starting materials react to form a product, according to a cascadeof elementary chemical reactions. See, A. Dömling in: MulticomponentReactions (J. Zhu, H. Bienayme) Wiley-VCH, Weinheim 2005, p. 76; A.Doemling, Org. Chem. Highlights 2005, Apr. 5. URL:http://www.organic-chemistry.org/Highlights/2005/05April.shtm; Kolb etal., 2002 Tetrahedron Lett. 43:6897; Fayol et al., 2005, Org. Lett.7:239). Usually the multiple reagents are introduced individually (i.e.,different reactants do not enter the reactor through the same port atthe same time) but reagents can also be introduced in variouscombinations.

In a fourth, related embodiment, a product is generated by a series ofreactions that generate intermediate products, e.g.:

A+B→X

C+D→Y

X+Y→P

where A, B, C and D are reactants introduced into the reactor, and X, Yand P are produced in the reactor under reaction conditions.

Combinatorial and Parallel Syntheses

It will be apparent from this disclosure that chemical reaction circuitscan be used to in combinatorial chemical syntheses. For example, theproducts of a first series of reactions (in reactors A, B, C, and D) canbe transported in various combinations (e.g., AA, AB, AC, AD, BC, BD,CD) to second reactors for combinatorial synthesis. It will be apparentfrom this disclosure that chemical reaction circuits can be run inparallel on the same chip. Up to several hundreds of thousands CRCs canbe placed on a single chip so that multiple sequential chemicalprocesses can be run in parallel.

Removing Product or Reagents from a Reactor

A reaction product can be removed from a reactor by flushing thereaction with a solvent (such as water). For example, as illustrated inFIG. 8C and corresponding text, solvent (e.g., water) can be flowed intoa reactor to flush the product out through an open valve into a flowchannel to a reservoir or other component of the system.

The spatial relationship between flow channels (such as distributionmanifolds) and reactors can be selected to accelerate or direct fluidmovement within an isolated microfluidic environment. For example,curved inlet and outlet channels for the elution of product from thereaction chamber. See, for example, FIG. 13 which shows arrangement of afluid inlet and outlet facilitate efficient removal of [¹⁸F]FDG from areactor. The tangential inlet and outlet allow the water trajectory tofollow along the far wall of the reaction chamber ensuring completeproduct elution. When the eluent solution enters and leaves the chamberthrough channels perpendicular to the tangent a product may be leftbehind, or a significant amount of solvent is required to wash it out ofthe reactor. With the curved inlet and outlet the solution follows atrajectory along the far wall of the reaction chamber and allowscollection of product in a small volume of solvent. In addition, makingthe exit channel slightly narrower than the inlet allows buildup of backpressure inside the chamber during elution, which also increases theefficiency of product collection.

Thus, one way to collect product from a coin-shaped (for example)reaction chamber is by flushing the product out through the tangentialexit channel by introducing solvent (e.g., water) into the reactorthrough another tangential channel, which allows the flow to follow thetrajectory along the far wall of the chamber. Although 1-3 reactorvolumes of water seem enough (experimentally) for complete collection ofproduct, larger elution volumes may be used, especially if largervolumes facilitate off-chip manipulations.

In an alternative approach a reactor having a radiator such as is shownin FIG. 20 and described above is drained in a two-step process: First,closing off the exit from the radiator will send N₂ pressure into thereactor through the gas permeable membrane. This pressure should forcethe product out of the reaction chamber into the only open exit channel.If at the same time the mixer depicted in FIG. 20 is activated, thisshould aid in directing the product solution into the exit channel andout of the reactor leaving behind at most droplets (rather than regions)of product solution. The second step should collect those droplets ofresidual product by filling the reactor with solvent (e.g., water) andforcing it.

Synthetic Method Comprising Column

In one aspect, the invention provides a method for carrying out achemical reaction in an integrated microfluidic device using amicrofluidic separation column, as described above. In one embodiment,the method includes introducing a solution containing a first reactantinto the separation column, and adsorbing the first reactant to thestationary phase of the column, eluting the first reactant from thestationary phase, and introducing the first reactant into a reactor. Inan embodiment, a second reactant is introduced into the reactor before,after, or simultaneously with the first reactant. In an embodiment, themethod includes maintaining the reactor for a time and under conditionssufficient for the first reagent and the second reagent to react andproduce a first reaction product.

The reactant is first bound to the stationary phase of the column in abinding step and then eluted from the stationary phase of the column inan elution step prior to being introduced into the reactor. In oneembodiment, the microfluidic device includes a closed flow path definedby the separation column and one or more flow channel(s) and the bindingstep includes circulating a solution comprising the first or secondreactant through the column at least twice. In one embodiment, thewherein said microfluidic device includes a closed flow path defined bythe separation column and one or more flow channel(s) and the elutingstep includes circulating an elution solution through the column atleast twice.

Section 5: Exemplary Devices

As will be clear from the discussion above, the invention provides alarge variety of microfluidic devices useful for solvent exchange and/orcarrying out a chemical reaction or sequential reactions and/or carryingout other processes. Any number of combinations and arrangements of thecomponents described herein are encompassed and the practitioner guidedby this disclosure will be able to design and produce devices.

For illustration and not limitation, one exemplary microfluidic deviceincludes a reactor (as defined above) that does not form a closed pathand has a liquid capacity of from 5 microliter to 10 microliters.

For illustration and not limitation, one exemplary microfluidic deviceincludes a separation column with an immobile phase through which afluid can pass, said column having an inlet and an outlet; and one ormore flow channel(s) not comprising the solid phase; where the flowchannel(s) and separation column define a closed path. In oneembodiment, the device has a peristaltic pump capable of moving fluidthrough the closed path. In one embodiment the device includes a reactorconfigured to be in fluidic communication with one or more flowchannels. In one embodiment, the microfluidic device has a closed flowpath defined by the separation column and one or more flow channel(s).

For illustration and not limitation, one exemplary microfluidic devicecontains vent channels. In one embodiment, an exemplary microfluidicsystem of the invention includes a device containing vent channels and(i) a means to flow gas (e.g., air, nitrogen, argon, etc.) through thevent channels or (ii) a vacuum pump or other means for applying a vacuumthat is connected to a vent channel system of a device. In oneembodiment, the

In one embodiment, an exemplary device contains from 1 to 5 reactors. Inone embodiment, an exemplary device includes parallel paths thatsimultaneously produce multiple batches of the same or differentcompounds.

In one aspect, a device of the invention is sterilized (e.g., by heat,toxic vapor, or irradiation). In one embodiment, the device is providedin a sterilized, aseptic form and packaged to maintain sterility untiluse.

In one aspect, the invention provides a microfluidic device capable ofsynthesizing ¹⁸F-FDG from precursors in 16 minutes or less, optionally5:50 minutes or less.

In one aspect, the invention provides a device comprising a flow channeland sieve valves. In one aspect, the invention provides a devicecomprising a flow channel and at least a pair of sieve valves, with achromatographic stationary phase material (e.g., beads or resin) isdisposed in the channel between the sieve valves, and where the size ofthe chromatographic stationary phase material is of a size that isretained by the sieve valve. In some embodiments, the device alsoincludes a reactor, as described herein.

Section 6: Examples Example 1 Synthesis of [¹⁹F]FDG

The molecular imaging probe 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG)is a widely used radiopharmaceutical with over a million patient dosesproduced in 2004 for use in positron emission tomography (PET) imagingstudies. The short half-life of [¹⁸F]fluorine makes rapid synthesis ofdoses essential, and the synthetic process includes common stepsrequired in many chemical syntheses, including the preparation of otherradiolabeled molecular imaging probes. The nanogram mass of PETmolecular imaging probes administered to subjects is ideal forminiaturized architecture of integrated microfluidics. Thus, themulti-step synthesis of [¹⁸F]FDG (see Hamacher et al., 1986, J. NuclearMedicine 27:235) and other PET probes represents an interestingopportunity for integrated microfluidics chips.

The synthesis of [¹⁸F]FDG (3a) is based on 5 sequential chemicalprocesses (FIG. 2A): (i) concentration of the dilute [¹⁸F]fluoridesolution (1-10 ppm) obtained from the proton bombardment of [¹⁸O]waterwith a cyclotron; (ii) solvent exchange from water to dry acetonitrile;(iii) [¹⁸F]fluoride substitution of the D-mannose triflate precursor 1in dry acetonitrile; (iv) solvent exchange back to water; and (v) acidichydrolysis of the fluorinated intermediate 2a to obtain [¹⁸F]FDG (3a).Presently, [¹⁸F]FDG (3a) is routinely produced in about 50 min usingcommercial synthesizers (Padgett et al., 1989, Applied Radiation andIsotopes 40:433). These automated synthesizers have a physical size ofapproximately 80×60×40 cm, and can produce ˜10 to 100 doses in a singlerun. Inevitably, a considerable decrease in the radiochemical yield ofthe resulting probe must be tolerated because of the relatively shorthalf-life of [¹⁸F]fluorine (t_(1/2)=110 min). Obtaining high yields iseven more challenging for molecular imaging biomarkers labeled withother important positron emitting radioisotopes with shorter half-lives,such as ¹¹C (t_(1/2)=20 min) and ¹³N (t_(1/2)=10 min).

A microfluidic chemical reaction circuit (CRC; FIG. 2) capable ofexecuting the five chemical processes of the syntheses of both [¹⁸F]FDG(3a) and [¹⁹F]FDG (3b) within a nanoliter scale reaction vessel wasdesigned and fabricated. In initial experiments, [¹⁹F]FDG (3a) wasproduced on the CRC using a multiprocess synthesis. In an initial step,¹⁹F[Fluoride] was concentrated from solution. The concentration step wasdeveloped because the concentration of [¹⁸F]fluoride obtained from aproton-bombarded [¹⁸O]water is usually below 1 ppm, and performingfluorination reaction at such a low [¹⁸F]fluoride concentration is notfeasible.

A miniaturized anion exchange column (FIG. 3) in the microfluidic deviceto concentrate the [¹⁸F]fluoride solution to ˜100 ppm was prepared.Sieve valves (FIG. 3B) were created using a square-profiled fluidicchannel and a control membrane. Actuation of this membrane prohibits thepassage of large particles while still permitting the solution to passthrough the edges of the channel. Using these sieve valves to trap anionexchange beads, the anion exchange column (FIGS. 3C and D) was obtainedfor the concentration.

For the first process, concentration of dilute fluoride, a 5 ppm NaFsolution was loaded into the anion exchange column (FIG. 4A). Theloading rate (5.0 nL/sec) was controlled using a metering pump. Afterthe fluoride solution was loaded completely, a K₂CO₃ solution (0.25 M,18 nL) was introduced to fill the rectangular loop. The circulating pumpmodule was then turned on so that the K₂CO₃ solution (0.25 M, 18 nL)could loop through the column continuously to produce a concentrated KFsolution. Because the fluorination (process iii) of the D-mannosetriflate 1 requires anhydrous conditions, a digitally controlled hotplate was used to heat the CRC for removing water (process ii) from theconcentrated KF solution (FIG. 4B). To completely extrude any remainingmoisture, dry MeCN was loaded into the reaction loop and the CRC washeated again. Moisture and MeCN vapor can penetrate and escape thegas-permeable PDMS matrix. Once the CRC had cooled to room temperature,an anhydrous MeCN solution (40 nL) containing the D-mannose triflate 1(92 ng, limiting reagent) and Kryptofix 222 (364 ng) was introduced intothe ring-shaped reaction loop containing the dried KF. Thisheterogeneous reaction mixture was mixed inside the loop using thecirculating pump. During this step (process iii), the CRC was heated(100° C. for 30 s and then 120° C. for 50 s) to yield the fluorinatedintermediate 2b (FIG. 4C), as analyzed by GC-MS. This analysis indicatedthat the conversion yields for the fluorination process were 98%. Afterremoving MeCN by direct evaporation, 3 N HCl solution (40 nL) wasinjected into the CRC and the hydrolysis (FIG. 4D, processes iv and v)of the intermediate 2b was conducted at 60° C. to obtain [¹⁹F]FDG (3b),in >90% purity, according to GC-MS analysis. The PDMS materials werecompatible (Lee et al., 2003, Analytical Chemistry 75:6544) with MeCNand the entire synthesis was demonstrated on multiple chips.

Example 2 Synthesis of [¹⁸F]FDG

Radioactive [¹⁸F]FDG (3a) was produced in the CRC by starting fromradioactive [¹⁸F]fluoride obtained from proton-bombarded [¹⁸O]water. Inthis experiment, only 720 μCi of [¹⁸F]fluoride (limiting reagent) in ca.1 μL of [¹⁸O]water was used, in an automated fashion. Because of therelative high loading rate (ca. 65 nL/sec) applied, only 500 μCi of[¹⁸F]fluoride was trapped in the column; the subsequent chemical stepsto produce [¹⁸F]FDG (3a) were completed within 14 min to obtain 190 μCiproduct 3a having a radiochemical yield of 38% and a radiochemicalpurity of 97.6%, according to radio-TLC analysis (FIG. 10). Similarresults were observed across multiple runs.

Materials and Methods

Fabrication of the first generation CRC. The chip was fabricated usingmulti-layer soft lithography method. (McDonald et al., 2000,Electrophoresis 21:27; Unger et al. 2000, Science 288:113.) Twodifferent molds were first fabricated by photolithographic processes tocreate the fluidic channels and the control channels for actuating thevalues located in the respective layers of the PDMS-based CRC. The moldused to create the fluidic channels was made by a following two-stepphotolithographic process. In the first step, a 45-μm thick negativephotoresist (SU8-2025) was spin coated on to a silicon wafer (SiliconQuest, San Jose, USA). After UV exposure and development, asquare-profiled pattern for the miniaturized anion exchange column wasobtained. In the next step, a second layer of 45-μm thick positivephotoresist (AZ 100XT PLP) was then spin coated on the same wafer. Priorto the UV exposure the mask was aligned (Karl Suss America Inc.,Waterbury, Vt.) to ensure a good match between two set of patterns ofthe control and fluid channels. Once the positive photoresist wasdeveloped, the wafer was heated above the glass transition temperatureof the positive photoresist. As a result, the surface profile of thepatterned positive photoresist was transformed into a round profilewhile the profile of the negative photoresist remains unchanged (squareprofile). This device has a channel height of 45 μm and width of 200 μm.The control channels mold was made by introducing a 25 μm-thin negativephotoresist (SU8-2025) pattern on a silicon wafer. In order to achievereliable performance of each valve, the width of the control channel wasset at 250 μm in sections where the valve modules are located.

Before fabricating the device, both the fluidic and control molds wereexposed to trimethylchlorosilane (TMSCl) vapor for 2-3 minutes. Awell-mixed PDMS (GE, RTV 615 A and B in 5 to 1 ratio) was poured ontothe fluidic mold located in a petri dish to give a 5 mm-thick fluidiclayer. Another portion of PDMS (GE, RTV 615 A and B in 20:1 ratio) wasspin-coated onto the control mold (1600 rpm, 60 s, ramp 15 s) to obtainthe control layer. The thick fluidic layer and thin control layer werecured in an 80° C. oven for 50 minutes. After incubation, the thickfluidic layer was peeled off the mold, and holes were introduced ontothe fluidic layer for access of reaction solutions. The fluidic layerwas then trimmed, cleaned and aligned onto the thin control layer. Afterbaking at 80° C. for 60 minutes, the assembled layer was peeled off thecontrol mold, and another set of holes were punched for access ofcontrol channels. These assembled layers were then placed on top of aglass slide that was coated (1600 rpm, 60 s, ramp 15 s) with PDMS (GERTV 615 A and B in 20:1 ratio) that had been cured for 45 minutes in theoven. The device was done after overnight incubation.

Control Interface. The pneumatic control setup consists of 4 sets ofeight-channel manifolds controlled through BOB3 breakout controllerboard (Fluidigm, San Francisco, USA). Argon gas that was pre-driedthrough a gas purifier (Hammond Drierit, Xenia, USA) provides pressure(30 psi) to the manifolds. 32 Control lines from the device areindividually connected to the corresponding channels on the manifoldswith metal pins (23 Gauge, New England Small Pin Corp, USA) using Tygonmicrobore tubing (Cole-Parmer East, Bunker Court, USA). When a channelon the manifold is activated, argon gas enters the control lineconnected with the specific channel, providing pressure to close valvesin the microfluidic device. The control interface was created usingLabview program on a PC. A National Instruments card (AT-DIO-32HS)digitally controls the switching of manifolds through the BOB3 breakoutcontroller board. The Labview program allows for manual control ofindividual valves and for automation of the synthesis processes.

Materials. All reagents were purchased from SIGMA-ALDRICH. Solventspurchased form VWR/EMD were purified according to literature procedure.Armarego et al., 2003, Purification of Laboratory Chemicals (ButterworthHeinemann, New York, ed. Fifth, 2003). GC-MS was performed with GC/EITime-of-Flight mass spectrometer (Micromass GCT). DBS-MS capillarycolumn (40 m long, 320 μm of OD) was employed for GC analyses of [¹⁹]FDGintermediate (2b) and product (3b) using Helium as carrier gas at flowrate of 1.2 mL/min. No-carrier-added [¹⁸F]fluoride (specificactivity: >10,000 Ci/mmol) was produced by 11 MeV proton bombardment of95% ¹⁸O-enriched H₂O via ¹⁸O(p,n)¹⁸F nuclear reaction using a RDS-112cyclotron. HPLC analysis was performed using a Rainin-HP system equippedwith a γ-detector. A Phenomenex column (Econosphere-NH₂, 5 μm, 250×4.6mm) was used with a solvent system of 85% MeCN and 15% H₂O. Radio-TLCanalysis was performed on silica plate (EM Separation Technology, Silicagel 60) with eluent system of 85% MeCN and 15% H₂O.

Preparation and Evaluation of Anion Exchange Column. Anion exchangebeads (Source 15Q, Amersham Biosciences) were packed into the columnmodule by introducing an aqueous solution containing suspended beadsinto the microfluidic reactor. The beads were activated by passing 1.0 Mof KHCO₃ through the column followed by sequential introduction of DIwater (18 MΩ).

Snapshots of FDG Synthesis in the CRC. The FDG (3a,b) synthesis in theCRC is based on three sequential synthetic processes starting from (i)concentration of dilute fluoride, followed by (ii) fluorine substitutionreaction of the D-mannose triflate precursor 1 and (iii) acidichydrolysis of the fluorinated intermediate 2a (or 2b). There were 15steps to complete the FDG (3a,b) synthesis in a CRC. The details ofthese sequential operations using schematic diagrams shown in FIGS. 6-8.

GC-MS analysis of the fluorinated intermediate 2b and [¹⁹F]FDG (3b). Thefluorinated intermediate 2b produced in the CRC was analyzed by GC-MS,indicating that the conversion yield for the fluorination reaction wasabout 95%. (FIG. 8B) Due to the low volatility of [¹⁹F]FDG (3b), the[¹⁹F]FDG (3b) obtained in the CRC had to be first treated by TMSCl priorto the GC-MS analysis. The GC-MS result indicated that the hydrolyticreaction of intermediate 2b results [¹⁹F]FDG (3b) in >90% purity. (FIG.9C)

1,3,4,6-Tetra-O-acetyl-2-[¹⁹F]fluoro-2-deoxy-D-glucose (2b). A 40 nLanhydrous MeCN solution containing1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulfonyl-D-manno-pyranose(mannose triflate) (1) (92 ng, 1.9×10⁻¹⁰ mol) and4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane (Kryptofix222) (364 ng, 9.6×10⁻¹⁰ mol) was introduced into the reaction loopcontaining the dried KF which was concentrated by the previous fluorideconcentration process. After all the valves around the reaction loopwere closed, the CRC was heated at 100° C. for 30 seconds and maintainedat 120° C. for 50 seconds. Meanwhile, the circulating pump was turned onto provide efficient mixing. After the chip was cooled down to 35° C.,the reaction residue inside the loop was flushed out with MeCN for GC-MSanalysis. Although an accurate reaction yield from GC-MS analysis couldnot be obtained it was obvious that the entire D-mannose triflateprecursor 1 disappeared after the reaction and the fluorinatedintermediate 2b was the only reaction product. (FIG. 9B)

2-Deoxy-2-[¹⁹F]fluoro-D-glucose([¹⁹F]FDG) (3b). After the fluorinationstep, 40 nL of HCl solution (3.0 N) was loaded into reaction loop. Withall the valves closed and circulating pump running, the hydrolysis ofthe fluorinated intermediate 2 was finished in 1 minute at a temperatureof 60° C. After cooling down to 35° C., the final product, [¹⁹F]FDG (3b)was flushed out from the CRC by water. Final aqueous solution wasremoved in vacuo. Due to the low volatility, [¹⁹F]FDG (3b) wasderivatized with TMSCl prior to GC-MS analysis. The GC-MS resultindicated that the hydrolytic reaction of intermediate 2b results[¹⁹F]FDG (3b) in >90% purity. (FIG. 9C)

2-Deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) (3a). 720 μCi of[¹⁸F]fluoride in ca. 1 μL of [¹⁸O]water was introduced into the fluorideconcentration loop of the CRC. Because a relatively high loading rate(65 nL/sec) was applied, only 500 μCi of [¹⁸F]fluoride (ca. 3.3×10⁻¹¹mol, limiting reagent) was trapped in the column. An 18 nL of K₂CO₃solution (0.25 M, 4.5×10⁻⁹ mole) was introduced to fill the rectangularloop, and the circulating pump module was then turned on so that theK₂CO₃ solution could loop through the column continuously to produce aconcentrated [¹⁸F]KF solution. After circulation, 20 nL of K₂CO₃solution was introduced into fluoride concentration loop to displace theconcentrated [¹⁸F]fluoride solution into the ring-shaped reaction loop.With all the valves around reaction loop closed, the CRC was heated on adigitally controlled hotplate. The CRC was cooled down to 35° C. within1 minute, and anhydrous MeCN (40 nL) was introduced into the reactionloop. The CRC was heated again to remove the water residue inside theloop. Kryptofix 222 (1.4 μg, 3.7×10⁻⁷ mol) and the mannose triflate 1(324 ng, 6.7×10⁻¹⁰ mol) in anhydrous MeCN were introduced into thereaction loop. The CRC was heated with a gradient (100° C./30 seconds,120° C./50 seconds). At the same time, the solution was actively mixedby the circulating pump to give [¹⁸F]fluorinated intermediate 2a in theCRC. After cooling the CRC down to 35° C. within 1 minute, HCl aqueoussolution (3.0 N) was introduced into the reaction loop. The mixture wasmixed by the circulating pump for 1 min at 60° C. In this step, theintermediate 2a was hydrolyzed to yield the final product [¹⁸F]FDG (3a).After cooling down to room temperature, the final product, 190 μCi of[¹⁸F]FDG (3a) (ca. 1.25×10⁻¹¹ mol, 38% yield) was flushed out from thechip by water for the analyses of radio-TLC (FIG. 10) and radio-HPLC.The analyses of radio-TLC and radio-HPLC suggested that the unpurifiedmixture obtained in the synthesis had a radiochemical purity of 97.6%.

Example 3 Second Generation CRC

This example describes a second generation CRC with the capacity tosynthesize larger [¹⁸F]FDG (3a) doses. This chip has a coin-shapedreactor (5 μL volume) equipped with a vacuum vent. It was used tosynthesize 1.74 mCi [¹⁸F]FDG (3a) sufficient for several mouse imagingexperiments. From the purified and sterilized product (FIG. 5A), twodoses (375 μCi and 272 μCi) were used for microPET- and microCT-basedmolecular imaging of two mouse models of cancer. Devices of this typecan be used to synthesizing PET imaging agents on 100 mCi scale.

Fabrication of the second generation CRCs. The second generation CRCs(FIG. 11) were manufactured by a soft lithography method similar to theone described above. (Fabricated at Fluidigm Corp.) The main differencesare (i) a third vent layer is located above the fluidic and controllayers, (ii) all three layers of the CRC were made from 10:1 A/B PDMSand are held together by placing a 1-μm thick layer of PDMS B componentbetween every two layers and (iii) the assembled device was mounted on a2-inch silicon wafer. The dimensions of the arched flow channels are250-μm wide and 45-μm tall. Only the water inlet channel is 300 μm wide.The reactor is 5 mm in diameter and 250 μm in height (with a totalvolume of 5 μL). The control channels form 250×250 μm intersections withflow channels. The vent is in the third layer with channels located 50μm above the reactor 250 μm apart from each other and measuring 250×250μm in cross-section.

Chip II Description and Logic: In order to increase the amount of[¹⁸F]FDG (3a) produced by a CRC to human dosage levels, the secondgeneration CRCs were developed. Key features include (i) a 5-μLcoin-shaped reactor, (ii) an overlaying vent channel connected to anexternal vacuum line, (iii) an external ion exchange column controlledby valves on the CRC, and (iv) a manifold for introducing the mannosetriflate. The geometry of reactor inlets/outlets determines the fluiddynamics inside the CRC leading to better mixing during the reaction.The architecture of the new CRC also minimizes the number of valves.Currently the largest [¹⁸F]FDG (3a) dose produced by this chip in asingle run equals 1.74 mCi. This chip was used to generate [¹⁸F]FDG (3a)for the mouse image presented in FIG. 12. The vacuum vent isadvantageous during the solvent exchange steps. Full operation of thechip during [¹⁸F]FDG (3a) synthesis is described in FIG. 12. [¹⁸F]FDG(3a) is eluted as an acidic solution with 96% purity, which is firstneutralized by 1.0 M NaHCO₃ and then passed through an alumina column(190 mg) to remove residual fluoride. The resulting solution exhibits99% [¹⁸F]FDG (3a) purity according to radio-TLC (FIG. 5A).

CRC II: Off-Chip Fluoride Concentration. For the operation of the newgeneration CRC, The [¹⁸F]fluoride concentration was carried using theautomatic Explora RN Nucleophilic [¹⁸F]Fluorination system (SiemensBiomarker Solutions, Culver City, Calif.). An MP-1 resin cartridge wasused to trap the dilute fluoride obtained from cyclotron. The activitywas eluted with a solution of K₂CO₃ (3 mg) in water (400 μL). Upon waterevaporation, the solution of Kryptofix 222 (20 mg) in MeCN (400 μL) wasadded followed by solvent evaporation. The residue was dissolved inanhydrous MeCN (200 μL) and the resulting solution of[¹⁸F]Fluoride/K₂CO₃/K222 in MeCN containing 700 mCi of [¹⁸F]fluoride wastransferred from the Explora system via approximately 2 m of tubing to aconical vial (source vial) located near the CRC. Application of 10 psiof pressure to the source vial introduced concentrated[¹⁸F]Fluoride/K₂CO₃/K222/MeCN mixture into the CRC.

Mouse Model. The tumor model used was a strongly immunogenic,non-metastasizing retrovirally-induced rhabdomyosarcoma (M-MSV/M-MuLV)(Fletcher et al., 2002, Tetrahedron 58:4735). MSV is areplication-defective, acutely transforming retrovirus carried withhelper activity provided by M-MuLV, which encodes the gag, pol, and envcomponents that are necessary for cell infection and replication (Worzet al., 2001, Chemical Engineering Science 56:1029). Rhabdomyosarcomasdevelop at the intramuscular inoculation site after a short latencyperiod (7-10 days) and regress over a period of 4-5 wk following theinduction of a strong immune reaction in immunocompetent adult mice.These lesions were characterized by a mixture of virus-infected myocytesand a large infiltrate of lymphocytes, granulocytes, and macrophages.Both cellular and humoral immune responses induced are dependent onpresentation by H-2 D^(b) alleles (Watts et al., 2003, Current Opinionin Chemical Biology 7:380). Rejection was mediated by CD8+ cytolytic Tcells, which recognize peptides from the gag and env proteins of M-MuLV,and requires help from CD4+ T cells (Kobayashi et al., 2004, Science304:1305; Chan et al., 2003, Nano Letters 3:199; Kawaguchi et al., 2005,Angewandte Chemie-International Edition 44:2413)

Mouse imaging. The tumor bearing mouse was injected with 272 microCi ofFDG via tail vein. Following 1-h uptake and non-specific clearance, themouse was imaged for 15 minutes in a Focus 220 microPET, followed by amicroCT scan (Siemens, Knoxville, Tenn.). MicroPET and microCT imageswere reconstructed using MAP and Fledkamp to resolutions of 1.2 mm and0.4 mm respectively, then fused using AMIDE image visualizationsoftware. See FIG. 5B for result.

Example 4 SYNTHESIS OF2-(1-(6-[(2-[¹⁸F]FLUORO-ETHYL)(METHYL)AMINO]-2-NAPHTHYL)(FDDNP)

This example describes the synthesis of2-(1-(6-[(2-[¹⁸F]fluoro-ethyl)(methyl)amino]-2-naphthyl)(FDDNP), amolecular imaging probe utilized in diagnosis of Alzheimer's disease.See, Agdeppa et. al., 2003, Mol. Imag. Biol. 5:404. The chemical stepsto produce [¹⁸F]FDDNP were completed within 20 minutes and resultedin >8 μCi product. Similar results were observed across multiple runs.

The chip design for FDDNP synthesis included a loop-type reactor,without an anion exchange column (see FIG. 14 and accompanying legend).The fabrication of the CRC for synthesis of [¹⁸F]FDDNP was generally asdescribed above in Example 2. All reagents with the exception of2-(1-{6-[(2-(p-Toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrile(provided courtesy of MTI) were purchased from SIGMA-ALDRICH.[¹⁸F]fluoride (limiting reagent) in about 1 μL of [¹⁸O]water was used inthe synthesis. The Kryptofix 222 plus2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrile(“precursor”) solution is made by adding 10 mg Kryptofix and 14micrograms precursor to 100 microliters anhydrous MeCN. A four-foldhigher concentration of precursor has also been used, with similarresults.

The FDDNP synthesis in the CRC is based on two sequential syntheticprocesses starting from (i) exchange of solvent from water toacetonitrile of dilute fluoride, followed by (ii) fluorine substitutionreaction of the2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrile.

For the synthesis, 200 μCi of [¹⁸F]fluoride in about 1 μL of [¹⁸O]waterplus K₂CO₃ (0.05 M) was introduced into the CRC. Because of the limitedvolume (350 nL) inside the reaction chamber, only 70 μCi of[¹⁸F]fluoride (ca. 4.6×10⁻¹² mol, limiting reagent) was inside thereaction loop. With all the valves around reaction loop closed, the CRCwas heated on a digitally controlled hotplate to evaporate the water.The CRC was cooled down to 35° C. within 1 minute, and anhydrous MeCN(350 nL) was introduced into the reaction loop. The CRC was heated againto remove the water residue inside the loop. Kryptofix 222 and2-(1-{6-[(2-(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-napthyl}ethylidine)malononitrilein anhydrous MeCN were introduced into the reaction loop. The CRC washeated with a gradient (100° C./30 seconds, 120° C./180 seconds). At thesame time, the solution was actively mixed by the circulating pump togive the final product. After cooling the CRC down to room temperaturewithin 1 minute, anhydrous MeCN was introduced into the reaction loopand actively mixed by the circulation pumps to dissolve and wash off anyproduct that might have attached to the wall of the reaction chamber.Lastly, anhydrous MeCN was used to flush the product off the chip forthe analyses of radio-TLC. Analyses of radio-TLC showed that theunpurified mixture obtained contained [¹⁸F]FDDNP at a radiochemicalpurity of 11.6%.

Example 5 Design, Fabrication and Use of a Microfluidic Device

This Example describes an early synthesis of FDG using a microfluidicdevice.

CRC Fabrication

The three-layer PDMS-based integrated CRCs were built using softlithography method (Liu et al., 2003, “Solving the “world-to-chip”interface problem with a microfluidic matrix” Analytical Chemistry75:4718-23. Two different molds were first fabricated byphotolithographic processes for production of fluidic channels and thecontrol channels located in each layer of the PDMS-based CRC.

The mold applied for fabrication of fluidic channels is fabricated by atwo-step photolithographic process. In principle, the thickness ofphotoresist determines the height of the fluidic/control channels.During the first step a 45-μm thick negative photoresist (SU8-2025) wasspin coated on to a silicon wafer. After UV exposure and development asquare-profiled pattern for a column module was obtained. In thefollowing step, a second layer of 45 μm thick positive photoresist (AZ100XT PLP) was then spin coated on the same wafer. An alignment processwas performed prior of the UV exposure to ensure a good match betweenthe first and second layers of fluidic channel. Once the positivephotoresist was developed, the wafer was heated above the glasstransition temperature of positive photoresist's. As a result, thesurface profile of the patterned positive photoresist was transform intoa round profile while the profile of the negative photoresist remainsintact. It is important to note that the heights and widths of thecolumn and fluidic channels have to be well-matched. For this device, achannel height of 45 μm and width of 200 μm is used. Thedimensionalities of column and fluidic channels were determined on thebasis of reaction volume and flow rate.

Mold applied for the control channels is made by introducing a 25μm-thin negative photoresist (SU8-2025) pattern on a silicon wafer. Theheight of the control layer should be above one tenth of the width ofcontrol channel to avoid collapses of valves during the CRC fabrication.The width of the control channels is very flexible. In order to achievereliable performance of each valve, the width of the control channel wasset to be 250 μm at the certain sections where the valve modules arelocated. To avoid parasitic valves from forming, width of the controlchannel are kept between 25 μm to 50 μm in all other areas. The width ofcontrol layer is inversely related to actuation pressure of the valves(Studer et al., 2004, “Scaling properties of a low-actuation pressuremicrofluidic valve.” J. Applied Physics 95:393-398).

Before fabricating the device, both the fluidic and control molds areexposed to vapor of trimethylchlorosilane (TMSCl) for 2-3 minutes. Awell-mixed PDMS (GE RTV 615 A and B in 5 to 1 ratio) was then pouredonto the fluidic mold located in a petri dish to produce a 5 mm-thickfluidic layer. Another portion of PDMS (GE RTV 615 A and B in 20:1ratio) is spin-coated onto the control mold (1600 rpm, 60 s, ramp 15 s)to give a control layer. The thick fluid layer and thin control layerare cured in an 80° C. oven for 150 minutes and 50 minutes,respectively. After incubation, the thick fluidic layer is peeled offthe mold, and holes were introduced onto the fluidic layer for access ofreaction solutions. This layer is then trimmed, cleaned and aligned ontothe thin control layer. After backing at 80° C. for another 90 minutes,the assembled layer was peeled off the control mold, and: another set ofholes are punched for access of control channels. Finally, thisassembled layer is then placed on top of a glass slide that is coatedwith a 20:1 ratio of GE RTV 615 A and B (1600 rpm, 60 s, ramp 15 s) thathad been cured for 45 minutes in the oven. The device was ready for useafter a oven overnight incubation in the oven. FIG. 15 shows aPDMS-based chemical reaction circuits for FDG synthesis.

FDG Synthesis

Three sequential synthetic processes of FDG in integrated CRCs. Similarto the conventional synthetic processes utilized for a laboratoryautomation system, FDG can be produced in the integrated CRC by threesequential steps, including (i) concentration of fluoride, (ii)fluorination and (iii) hydrolysis.

(1) Concentration of dilute fluoride: By using an anion exchange column(FIG. 13) dilute fluoride (1 ppm with the volume raging form 10 to 500μL) was concentrated in the square-shape mixer to obtain a three to fourorders more concentrate KF solution with a volume of 45 nL.

In order to perform the concentration of dilute fluoride (first step ofFDG synthesis), a column module capable of fluoride ion extraction wasincorporated into the CRC. An ion exchange column (FIG. 13) wasconstructed by trapping ion exchange beads in a fluid channel isolatedwith five sieve valves. A sieve valve composed of a square-profilefluidic line and a regular control membrane is different from a normalvalve based on a round-profile fluidic line (see FIGS. 3A and B). Ingeneral, when valves operate, the valve membranes deflect in an ellipticshape. In the case of normal valve, the deflected membrane is fullycompliant to the round-profile fluidic channel lead to complete close ofthe valve. For a sieve valve, a deflected membrane partially closes thevalve, generating two small gaps the two channel edges of thesquare-profile channel. When aqueous solution containing suspended beadsin appropriate sizes is introduced into the fluidic chambers, the beadsare trapped by the sieve valves while the solution is allowed to passthrough the closed sieve valve.

Source 15Q anion exchange resins (2 pm, Amersham Bioscience) was firstconfined inside the column module. A 1.0 M solution of KHCO₃ was thenpassed through the column to convert the resins into the saturatedbicarbonate forms. In order to remove the excess bicarbonate anions thatare not coupled with the quaternary ammonium groups on the beads surfacedeionized water was subsequently flown through the column before usedfor fluoride concentration.

A 200 μL aqueous solution containing cold fluoride (¹⁹F—) was passedthrough the ion exchange column. The negatively charged fluoride ionswere then exchanged onto the resin surfaces to replace HCO₃ counterions. After the fluoride solution has completely passed through, 45 nLof 0.02 M solution of K₂CO₃ was introduced into the first(square-shaped, FIG. 16A) reaction loop. The-pump module was then turnedon for two minutes so K₂CO₃ solution looped through the columncontinuously. As a result, the surface-trapped fluoride ions were elutedoff the column to a KF in solution which is ready to be transferred intothe reaction loop for further process.

(2) Fluorination (synthesis of1,3,4,6-tetra-O-acetyl-2-fluoro-2-deoxy-D-glucose): The synthesis of1,3,4,6-tetra-O-acetyl-2-fluoro=2-deoxy-D-glucose from1,3,4,6-tetra-O-acety Pl-2-trifluoro-methane-sulfonyl-D-manno-pyranose(mannose triflate) can be adapted from a previously developed method(Kikutani et al., 2004, Macromolecular Rapid Communications 25:158). Inthis case, mannose triflate was reacted with the complex of potassiumfluoride (KF) and4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane (Kryptofix222) in the anhydrous acetonitrile solution in the second (round-shaped,FIG. 16B) reaction chamber. Following the concentration of fluoride,water in the concentrated KF aqueous solution obtained from firstreaction chamber has to be removed. In the PDMS-based CRCs, removal ofwater was achieved by heating the CRCs directly on a hot plate at 120°C. for 2 minutes, whereupon all valves associated with the reaction loopwere closed. In order to ensure the complete removal of water residue,the loop was filled with dry acetonitrile and then heated to 150° C. for1 minute. It is important to note that moisture and acetonitrile vaporcan easily penetrate PDMS materials since the material is highlypermeable to gases. In a conventional automation system the removal ofwater in KF solution was carried out by heating the solution under anitrogen flow, which is less efficient and much more time-consuming whencompared with the same process in the PDMS-based CRC.

Immediately after the removal of water, 17 nL of acetonitrile solutioncontaining Kryptofix 222 (1.7 μg) and mannose initiate (272 ng) wasintroduced into the reaction loop from the top fluidic channel ofreaction loop presented in FIG. 16B. After closing all inlet/outletvalves, the inhomogeneous reaction mixture was then circulated insidethe reaction loop by using the peristaltic pump located on the up-leftportion of the loop; at the same time, the CRC was healed on a hotplateat 150° C. for 30 seconds. At this stage, the resulting intermediate wasanalyzed by a GC-MS. At the temperature covering 60 to 120° C., therespective yields of the fluorination process were obtained in the rangeof 0.6% to 16.5%. At 150° C., the GC-MS indicated that all the startingmaterial has been transformed after reaction time of 30 seconds.

(3) Hydrolysis (hydrolysis of1,3,4,6-tetra-O-acetyl-2-fluoro-2-deoxy-D-glucose to FOG): After thefluorination reaction, acetonitrile was removed by direct evaporationthrough PDMS materials prior to the following hydrolysis reaction. 17 nL6M. HCl solution was injected into the CRC, and the hydrolysis of1,3,4,6-tetra-O-acetyl-2-fluoro-2-deoxy-D-glucose was carried out by atroom temperature. Again, with the assistance of peristaltic pump thehydrolysis reaction was finished in 4 minutes. FIG. 17 summarizes theoverall three sequential reactions in the CRC. where fluorideconcentration, fluorination and hydrolysis are labeled in red, blue andgreen, respectively. In addition, the direction for introducingsolutions, beads and reagents are Indicated by arrows.

In a laboratory automation system, it takes about 40 minute to completethe processes of fluorination and hydrolysis for FDG synthesis. By usingour prototype CRC, these sequential synthetic processes can completedwithin 16 minutes, and we anticipate that the production time canshorten to 5 minutes or less when this CRC is fully automated. Aside-by-side comparison based on respective steps for a conventionalmethod and the CRC are outlined in Table 2.

Table 2 shows the reaction times and conditions of conventional andmicrofluidic preparations of FDG. FDG synthesis in microfluidic devicetook much less time than conventional method mainly due to the fasterreaction speed and quicker solvent evaporation (water and acetonitrile)in microfluidic chips. Multi-steps organic reactions can beautomatically finished step by step by controlling the pumps and valveswhich can be fabricated in the PDMS based chips.

TABLE 1 Conventional Method Microfluidic Time mg compounds in ml ngcompounds in (this Time solvent Time nL solvent example) (projected)Step Addition of K₂CO₃ in H₂O 0:04:00 Addition of 0.24 0:00:30  :00:20 1M K₂CO₃ to concentrating loop Step Addition of Kryptofix in 0:06:00elution of 18F off 0:02:00 0:01:00 2 MeCN and evaporation ion exchangecolumn Step Cooling of solution 0:00:30 Transfer of 0:02:00 0:01:00 3concentrated 18F into 2nd reaction loop Step Addition MeCN 0:04:00Evaporation of 0:05:00 0:02:00 4 H₂O and Addition of MeCN Step Additionof Triflate 0:05:00 Addition of triflate 0:02:00 0:00:30 5 suspended inMeCN and Kryptofix suspended in MeCN Step Fluorination of precursor0:04:00 Fluorination of 0:00:30 0:00:30 6 triflate heat it up to 150 C.°Step Hydrolysis (1M HCl) 0:16:00 Hydrolysis (6M 0:04:00 0:00:30 7 HCl)Total 39.30 16:00 5:50 Time

Example 6 Design, Fabrication and Use of a Microfluidic Device With anOff-Chip Column

FIG. 18 shows a chip with a coin-shaped reactor and off-chip anionexchange column. Valves are represented by red rectangles. Allinlet/outlet channels are the same size except for the larger inlet forthe water (product elution). The solution distributed through themanifold travels the same distance from the origin to the chamberthrough all 6 ports.

The dilute [F-18] solution is passed through the column and trapped bythe resin off-chip. This process is controlled by on-chip valves onshort channels connecting source of [F-18] to the column and the latterto the collection vial for H₂ ¹⁸O.

The aqueous K₂CO₃ solution routed through the chip and controlled byon-chip valves is then passed through the off-chip ion exchange columnto elute the [¹⁸F] straight into the reactor on the chip. At this pointwater can be evaporated leaving behind K¹⁸F and K₂CO₃ salts along withsome residual moisture. (The vacuum vent is used in this and allsubsequent evaporation steps. As a result the water vapor permanentlyleaves the chip rather than staying condensed in the chip's matrix. Inorder to remove this moisture by forming an azeotrope with MeCN andsolubilize K¹⁸F in organic solvens, the chamber is now filled with aMeCN solution of Kryptofix222, followed by its evaporation. At thispoint mannose triflate will be introduced into the reactor through a6-port manifold. Actuating the mixer at an elevated temperature shouldallow for efficient fluorination. It is also possible to achievefluorination at ambient temperature. Upon completion of thefluorination, the solvent is partially removed by evaporation. If MeCNis evaporated completely, [F-18]FTAG forms a thick oily residuedistributed unevenly throughout the reactor. This residue is verydifficult to dissolve in an aqueous solution in the next step. 3N HClsolution is introduced to the half-empty reactor through one channeluntil the reactor is full. Efficient mixing with MeCN solution of[F-18]FTAG is achieved quickly since it is facilitated at the interfaceof two solutions by swirling resulting from an acid-base reaction (withK₂CO₃). Heating at 60° C. followed by 75° C. allows hydrolysis toproceed to completion by gradual (but fast) removal of MeCN resulting inan aqueous solution of [F-18]FDG. Two ways for collecting the productfrom the reaction chamber after deprotection are proposed: 1) Flushingthe product out through the tangential exit channel by introducing waterinto the reactor through another tangential channel allows the flow tofollow the trajectory along the far wall of the chamber or removingproduct in a two-step process involving, first, closing off the exitfrom the radiator will send N₂ pressure into the reactor through the gaspermeable membrane and, second, activating the mixer shown in FIG. 19.

The product solution is delivered to a vial containing 2M KHCO₃ solutionto neutralize HCl. Afterwards the contents of the vial are passedthrough an alumina column resulting in 99.3% radio-pure [F-18]FDG.

Although the present invention has been described in detail withreference to specific embodiments, those of skill in the art willrecognize that modifications and improvements are within the scope andspirit of the invention, as set forth in the claims which follow. Allpublications and patent documents (patents, published patentapplications, and unpublished patent applications) cited herein areincorporated herein by reference as if each such publication or documentwas specifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same. Theinvention having now been described by way of written description andexample, those of skill in the art will recognize that the invention canbe practiced in a variety of embodiments and that the foregoingdescription and examples are for purposes of illustration and notlimitation of the following claims.

1. A method for solvent exchange using a microfluidic device, saidmethod comprising: i) providing a microfluidic device comprising areactor, wherein said reactor a) is configured to fluidicallycommunicate with at least one microfluidic channel; b) is configured tobe fluidically isolated; and c) is defined by a wall at least a portionof which is permeable to a gas but substantially impermeable to a liquidcorresponding to the gas; ii) introducing into the reactor a firstsolvent system comprising a first reactant; iii) fluidically isolatingthe reactor and withdrawing some or all of the first solvent system fromthe fluidically isolated reactor while retaining the first reactant inthe reactor; iv) introducing into the reactor a second solvent systemdifferent from the first solvent system.
 2. A method for removing asolvent system from a microfluidic reactor, said method comprising: i)providing a microfluidic device comprising a reactor, wherein saidreactor a) is configured to fluidically communicate with at least onemicrofluidic channel; b) is configured to be fluidically isolated; andc) is defined by a wall at least a portion of which is permeable to agas but substantially impermeable to a liquid corresponding to the gas;wherein said reactor optionally contains a first solvent system and, ifpresent, said first solvent system comprises a first solute andoptionally comprised additional solutes; ii) introducing into thereactor a second solvent system comprising a second solute andoptionally comprised additional solutes; iii) isolating the reactor,whereby the reactor contains a third solvent system and a solute denotedSolute A, wherein Solute A is first solute, the second solute, or aproduct of a reaction in which either or both of the first and secondsolutes are reactants and wherein said third solvent system is the sameas the second solvent system or is a solvent system comprised of thecombination of the first and second solvent systems; iv) withdrawing atleast 25% of the volume of the third solvent system from the fluidicallyisolated reactor, wherein the third solvent system is withdrawn from thereactor more rapidly than Solute A is withdrawn, and wherein the amountof Solute A in the reactor per unit volume of the third solvent systemin the reactor increases as the third solvent system is withdrawn. 3.The method of claim 2 wherein Solute A is in solution in the thirdsolvent system the concentration of Solute A in the reactor increases asthe third solvent system is withdrawn.
 4. wherein The method of claim 1wherein the second solvent system comprises a second reactant, andwherein the first reactant and the second reactant are compounds thatchemically react, under reaction conditions, to generate a firstreaction product.
 5. The method of claim 4 comprising the further stepsof fluidically isolating the reactor and maintaining the reactor in afluidically isolated state for a time and under conditions sufficientfor the first reaction product to accumulate in the reactor.
 6. Themethod of claim 5 comprising the further step of withdrawing some or allof the reaction solvent system from the fluidically isolated reactorwhile retaining the product in the reactor.
 7. A method for carrying outa chemical reaction using a microfluidic device, said method comprising:i) providing a microfluidic device comprising a reactor, wherein saidreactor a) is configured to fluidically communicate with at least onemicrofluidic channel; b) is configured to be fluidically isolated; andc) is defined by a wall at least a portion of which is permeable to agas but substantially impermeable to a liquid corresponding to the gas;ii) introducing into the reactor a first solvent system comprising afirst reactant; iii) introducing into the reactor a second solventsystem comprising a second reactant, wherein the first reactant and thesecond reactant are compounds that chemically react, under reactionconditions, to generate a product; iv) fluidically isolating thereactor, whereby the reactor contains 1) a reaction solvent system and2) the first and second reactants and/or the product.
 8. The method ofclaim 7 further comprising v) maintaining the reactor in a fluidicallyisolated state for a time and under conditions sufficient for a firstreaction product to accumulate in the reactor; vi) withdrawing some orall of the reaction solvent system from the fluidically isolated reactorwhile retaining the product in the reactor.
 9. The method of claim 5comprising i) fluidically joining the reactor and a microfluidicchannel; ii) introducing into the reactor a third solvent systemcomprising a third reactant and/or a catalyst, while retaining the firstproduct in the reactor; iii) maintaining the reactor in a fluidicallyisolated state for a time and under conditions sufficient for a secondreaction product to accumulate in the reactor. 10-11. (canceled)
 12. Themethod of claim 7 comprising i) providing a microfluidic devicecomprising a reactor wherein said reactor a) is configured tofluidically communicate with at least one microfluidic channel; b) isconfigured to be fluidically isolated; and c) is defined by a wall atleast a portion of which is permeable to a gas but substantiallyimpermeable to a liquid corresponding to the gas; ii) reacting a firstreactant and a second reactant in the reactor, wherein the first andsecond reactants are in solution in a reaction solvent system, whereinthe reactor is fluidically isolated, and wherein a first reactionproduct is produced; iii) evaporating at least a portion of the reactionsolvent system from the fluidically isolated reactor; iv) introducinginto the reactor a solution comprising a third reactant and/or acatalyst, while retaining the first product in the reactor.
 13. Themethod of claim 7 wherein the reactor is coin-shaped and/or wherein ventchannels are positioned adjacent over the reactor.
 14. The method ofclaim 7 wherein the first reactant or the second reactant is purified orconcentrated in an on-chip microfluidic separation column prior to beingintroduced into the reactor.
 15. The method of claim 7 wherein themicrofluidic device comprises a separation column comprising astationary phase; said method comprising: (i) introducing into theseparation column a solution containing the first reactant, andadsorbing the first reactant to the stationary phase; (ii) eluting thefirst reactant from the stationary phase in a solvent system; (iii)introducing said solvent system comprising said first reactant into thereactor.
 16. (canceled)
 17. A method for carrying out sequentialchemical reactions using an integrated microfluidic device comprising i)providing a microfluidic device comprising a reactor and providingreagents sufficient for carrying out at least two sequential chemicalreactions; ii) carrying out a first chemical reaction in the reactor,thereby producing a product; iii) carrying out a second chemicalreaction in the reactor, wherein the product from (ii) is a reactant inthe second chemical reaction and wherein the product from (ii) is notremoved from the reactor prior to step (iii). 18-34. (canceled)
 35. Amethod according to claim 17 for carrying out a series of chemicalreactions using a microfluidic device said method comprising i)providing a microfluidic device comprising a reactor, wherein saidreactor a) is configured to fluidically communicate with at least onemicrofluidic channel; b) is configured to be fluidically isolated; andc) is defined by a wall at least a portion of which is substantiallyimpermeable to liquid water and liquid acetonitrile, but permeable towater vapor and acetonitrile vapor; ii) introducing into the reactor anaqueous solution comprising [¹⁸F]fluoride; iii) introducing into thereactor an acetonitrile solution comprising mannose triflate; iv)fluidically isolating the reactor; v) reacting the [¹⁸F]fluoride and themannose triflate to produce2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose; vi)fluidically joining the reactor and a microfluidic channel; vii)introducing aqueous HCl into the reactor while retaining the2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose in thereactor; viii) fluidically isolating the reactor; ix) hydrolyzing the2-deoxy-2-¹⁸F-fluoro-1,3,4,6-tetra-O-acetyl-beta-D-glucose to produce¹⁸F-FDG. 36-38. (canceled)
 39. A microfluidic device that comprises: aseparation column comprising a solid phase through which a fluid canpass, said column having an inlet and an outlet; one or more flowchannel(s) not comprising the solid phase, wherein the flow channel(s)and separation column define a closed path; and a peristalic pumpcapable of moving fluid through the closed path. 40-41. (canceled)
 42. Amicrofluidic device comprising a reactor, wherein the reactor i) doesnot form a closed path; ii) is configured to be fluidically isolated;iii) has a liquid capacity of from 5 microliters to 10 microliterswherein the reactor is configured to fluidically communicate with atleast one microfluidic flow channel that is a distribution manifold iv)is defined by a wall at least a portion of which is permeable to a gasbut substantially impermeable to a liquid corresponding to the gas,wherein said liquid is selected from the group consisting of water,acetonitrile, and mixtures of water and acetonitrile.
 43. The device ofclaim 42 that contains from 1 to 5 reactors. 44-59. (canceled)
 60. Amethod for synthesizing a radiolabeled product in a microfluidicenvironment comprising mixing a radiolabled reactant with a precursorreactant compound to produce a radiolabeled product, wherein said mixingand reacting occurs in a microfluidic reactor and wherein theradiolabled reagent is introduced into the reactor in a first solventand the radiolabeled precursor is introduced in a second solvent that isdifferent from the first. 61-63. (canceled)
 64. The method of claim 60,further comprising deprotection or chemical modification of theradiolabeled product to produce a radiodiagnostic agent orradiotherapeutic agent.
 65. The method of claim 64 wherein theradiolabeled product is a radiolabeled molecular imaging probe.
 66. Themethod of claim 65 wherein the precursor reactant is D-mannose triflate;2-(1-{6-[(2-[(p-toluenesulfonyloxy)ethyl)(methyl)amino]-2-naphthyl}ethylidine)malononitrile;N-Boc-5′-O-dimethoxytrityl-3′-O-(4-nitrophenylsulfonyl)-thymidine;N2-(p-anisyldiphenylmethyl)-9-[(4-p-toluenes-ulfonyloxy)-3-(p-anisyldiphenylmethoxymethyl)butyl]guanine;N2-(p-anisyldiphenylmethyl)-9-[[1-[(.beta.-anisy-ldiphenylmethoxy)-3-(p-toluenesulfonyloxy)-2-propoxy]methyl]guanine;8-[4-(4-fluorophenyl)-4,4-(ethylenedioxy)bu-tyl]-3-[2′-(2,4,6-trimethylphenylsulfonyloxyethyl)]-1-phenyl-1,3,8-triazas-piro[4.5]decan-4-one;5′-O-Boc-2,3′anhydrothymidine;N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-1]-4-nitro-N-2-pyridinyl-benzamide;1,2-bis(tosyloxy)ethane and N,N-dimethylethanolamine; ditosylmethane orN,N-dimethylethanolamine.
 67. (canceled)